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Defining the molecular mechanisms of subtype-specific KCNQ2/3 potassium channel activators Wang, Wei-Ting 2016

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	THE	FACULTY	OF	GRADUATE	AND	POSTDOCTORAL	STUDIES		(Pharmacology)		THE	UNIVERSITY	OF	BRITISH	COLUMBIA	(Vancouver)		April	2016		©	Wei-Ting	Wang,	2016		 			 ii	Abstract	Retigabine	is	the	first	approved	anti-epileptic	drug	that	acts	via	activation	of	voltage-gated	 potassium	 channels,	 targeting	 KCNQ	 channels	 that	 underlie	 the	neuronal	M-current.	Retigabine	exhibits	little	specificity	between	KCNQ2-5,	which	all	contain	a	Trp	residue	in	the	pore	domain	that	is	essential	for	retigabine	actions.	The	retigabine	 analog	 ICA	 069673	 (‘ICA73’)	 exhibits	 much	 stronger	 effects	 than	retigabine	on	KCNQ2	channels,	including	a	large	hyperpolarizing	shift	of	the	voltage-dependence	of	activation,	and	roughly	two-fold	enhancement	of	peak	current.	Unlike	retigabine,	 ICA73	 exhibits	 strong	 subtype	 specificity	 for	 KCNQ2	 over	 KCNQ3,	 and	appears	to	have	a	unique	mechanism	of	action,	because	pore	mutations	that	abolish	retigabine	action	(KCNQ2	Trp236Phe)	do	not	affect	ICA73	sensitivity.	Based	on	ICA73	sensitivity	 of	 chimeric	 constructs	 of	 the	 transmembrane	 segments	 of	 KCNQ2	 and	KCNQ3,	this	drug	appears	to	interact	with	the	KCNQ2	voltage	sensor	(S1-S4)	rather	than	 the	 pore	 region	 targeted	 by	 retigabine.	 KCNQ2	 point	mutants	 in	 the	 voltage	sensor	were	generated	based	on	KCNQ2/KCNQ3	sequence	differences,	and	screened	for	 ICA73	 sensitivity.	 These	 experiments	 reveal	 that	 KCNQ2	 residues	 Phe168	 and	Ala181	 in	 the	S3	 segment	are	essential	determinants	of	 ICA73	subtype	 specificity.	Mutations	at	either	position	in	KCNQ2	abolish	the	ICA73-mediated	gating	shift,	while	retaining	retigabine	sensitivity.	Interestingly,	KCNQ2[A181P]	mutant	channels	show	little	ICA73-mediated	gating	shift,	but	retain	current	potentiation	by	the	drug.	When	Phe168	 and	 Ala181	 are	 substituted	 into	 KCNQ3	 ([L198F]	 and	 [P211A]),	 ICA73-sensitivity	can	be	partially	rescued.	These	results	demonstrate	 that	retigabine	and			 iii	ICA73	 act	 via	 distinct	 mechanisms,	 and	 provide	 the	 first	 insights	 into	 channel	residues	 that	 underlie	 subtype	 specificity	 of	 KCNQ	 channel	 openers.	 Further	mutagenic	scanning	of	the	voltage	sensor,	and	screening	for	potential	ICA73	binding	residues	in	solvent	accessible	pockets	have	also	generated	new	insights	into	KCNQ	channel	 function,	 despite	 not	 identifying	 additional	 residues	 essential	 for	 ICA73	sensitivity.	Taken	together,	findings	presented	in	this	thesis	have	laid	a	foundation	for	 further	 understanding	 of	 diverse	 mechanisms	 of	 action	 of	 KCNQ	 potassium	channel	openers,	which	may	lead	to	more	targeted	and	rational	approaches	for	drug	design.			 			 iv	Preface		 I	conducted	the	majority	of	the	experiments	and	analyzed	the	data	presented	in	 this	 thesis.	 Several	 constructs	 generated	 were	 through	 the	 help	 of	 our	 lab	technician,	Dr.	Runying	Yang.	 	Data	presented	in	Chapter	3	has	been	submitted	for	publication	in	April	2016,	while	data	presented	in	Chapter	4	is	part	of	several	ongoing	studies	related	to	KCNQ	openers	in	Dr.	Kurata’s	lab.		 			 v	Table	of	Contents	Abstract	........................................................................................................................................................................	ii	Preface	..........................................................................................................................................................................	iv	Table	of	Contents	....................................................................................................................................................	v	List	of	Figures	..........................................................................................................................................................	vii	List	of	Abbreviations	.........................................................................................................................................	viii	Acknowledgments	.................................................................................................................................................	ix	Chapter	1:	Introduction	and	Background	.................................................................................................	1	1.1	Kv	Overview	.......................................................................................................................	1	1.1.1	Overall	Structure	of	Kv	Channels	..............................................................................................	2	1.1.2	Ion	Selectivity	of	Kv	Channels	.....................................................................................................	2	1.1.3	Kv	Channel	Gating	and	Voltage-Sensing	...............................................................................	4	1.2	KCNQ	Overview	.................................................................................................................	5	1.2.1	M-current	...............................................................................................................................................	7	1.2.2	KCNQ	Channels	and	Disease	.......................................................................................................	8	1.3	PIP2	Regulation	of	KCNQ	................................................................................................	15	1.3.1	PIP2	.........................................................................................................................................................	15	1.3.2	PIP2	Effects	on	KCNQ	Channels	...............................................................................................	16	1.3.3	Potential	PIP2	Binding	Sites	.....................................................................................................	16	1.4	KCNQ	Channel	Openers	..................................................................................................	17	1.4.1	Development	of	RTG	.....................................................................................................................	18	1.4.2	Other	KCNQ	Openers	....................................................................................................................	20	1.5	Overview	of	Chapter	3:	Sequence	Determinants	of	Subtype-specific	Actions	of	KCNQ	Channel	Openers	.........................................................................................................	21	1.6.	Overview	of	Chapter	4:	In-depth	Analysis	of	Voltage	Sensor	Residues	Involved	in	ICA73	Effects	on	KCNQ2	.........................................................................................................	22	Chapter	2:	Materials	and	Methods	.........................................................................................	24	2.1	KCNQ2	and	KCNQ3	Channel	Constructs	......................................................................	24	2.2	Cell	Culture	and	Whole-cell	Patch	Clamp	Recordings	..............................................	25	2.3	Two-electrode	Voltage	Clamp	Recordings	.................................................................	25	2.4	Non-radioactive	Rb+	Efflux	Assay	.................................................................................	26	2.5	Data	Analysis	...................................................................................................................	27	Chapter	3:	Sequence	Determinants	of	Subtype-specific	Actions	of	KCNQ	Channel	Openers	.......................................................................................................................................	28			 vi	3.1	Introduction	.....................................................................................................................	28	3.2	Results	...............................................................................................................................	30	3.2.1	Subtype-specific	Effects	of	ICA069673	..............................................................................	30	3.2.2	Potentiation	and	Shift	in	Voltage-dependent	Gating	of	KCNQ2	...........................	32	3.2.3	ICA73	Acts	within	the	VSD	.........................................................................................................	33	3.2.4	Identification	of	Amino	Acid	Determinants	of	ICA73	Subtype	Specificity	....	36	3.2.5	Aromatic	Residues	at	KCNQ2	Residue	168	Preserve	ICA73	Sensitivity	.........	38	3.2.6	Proline	at	Position	181	in	KCNQ2	Disrupts	ICA73	Effects	......................................	40	3.2.7	ICA73-insensitive	Mutations	Preserve	Retigabine	Sensitivity	............................	42	3.2.8	ICA73-sensitivity	can	be	Transferred	to	KCNQ3	..........................................................	43	3.3	Discussion	........................................................................................................................	44	Chapter	4:	In-depth	Analysis	of	Voltage	Sensor	Residues	Involved	in	ICA73	Effects	on	KCNQ2	..........................................................................................................................................	49	4.1	Introduction	.....................................................................................................................	49	4.2	Results	...............................................................................................................................	51	4.2.1	Characterizing	the	Influence	of	the	S1-S2	Segments	on	ICA73	Sensitivity	...	51	4.2.2	ICA73	Effect	on	S1	Point	Mutations	.....................................................................................	52	4.2.3	ICA73	Effect	on	S2	Point	Mutations	.....................................................................................	54	4.2.4	Potential	Binding	Pocket	Residues	and	ICA73	Effects	..............................................	56	4.2.5	KCNQ2	I115F	may	have	Reduced	Current	Potentiation	by	ICA73	.....................	58	4.2.6	KCNQ2[E130A]	Abolishes	the	ICA73-mediated	Shift	but	not	Current	Potentiation	...................................................................................................................................................	60	4.2.7	KCNQ2	K219	may	Serve	Important	Role	in	Channel	Opening	..............................	62	4.3	Discussion	........................................................................................................................	64	Chapter	5:	General	Discussion	................................................................................................	67	References	..................................................................................................................................	76		 			 			 vii	List	of	Figures	Figure	3-1	ICA069673	exhibits	subtype-specificity	for	KCNQ2	over	KCNQ3	channels..........................................................................................................................................................................	31	Figure	3-2	ICA73	potentiates	KCNQ2	currents	and	induces	a	large	hyperpolarizing	shift	of	the	conductance-voltage	relationship.	...........................................................................	33	Figure	3-3	Substitution	of	KCNQ3	VSD	into	KCNQ2	alters	channel	sensitivity	to	ICA73.	...........................................................................................................................................................	35	Figure	3-4	Rubidium	efflux	screen	identifies	two	S3	mutations	that	diminish	ICA73	response.	.....................................................................................................................................................	37	Figure	3-5	Functional	characterization	of	KCNQ2[F168L]	mutant	channels.	..............	39	Figure	3-6	Functional	characterization	of	KCNQ2[A181P]	mutant	channels	illustrates	the	separable	nature	of	current	potentiation	and	gating	shift.	.....................	41	Figure	3-7	ICA73-insensitive	mutants	do	not	perturb	retigabine	sensitivity.	.............	43	Figure	3-8	ICA73	effect	can	be	partially	rescued	by	substitution	of	F168	and	A181	into	KCNQ3.	...............................................................................................................................................	44	Figure	4-1	S1-S2	domain	and	ICA73	sensitivity.	.......................................................................	52	Figure	4-2	Characterizing	S1	point	mutations	and	ICA73	effect.	.......................................	53	Figure	4-3	Characterizing	S2	point	mutations	and	ICA73	effect.	.......................................	55	Figure	4-4	ICA73	effect	on	potential	binding	pocket	residues.	..........................................	57	Figure	4-5	Current	potentiation	by	ICA73	may	be	reduced	in	KCNQ2[I115F].	...........	59	Figure	4-6	ICA73-mediated	shift	is	abolished	in	KCNQ2[E130A]	which	preserves	current	potentiation.	.............................................................................................................................	61	Figure	4-7	Examining	the	potential	role	of	KCNQ2	K219	in	channel	opening.	............	63			 viii	List	of	Abbreviations	AIS	 	 	 	 axon	initial	segment	PIP2		 	 	 	 phosphatidylinositol	4,5-bisphosphate	VSD		 	 	 	 voltage-sensing	domain	PGD		 	 	 	 pore-gating	domain	LQTS	 	 	 	 long	QT	syndrome	SQTS	 	 	 	 short	QT	syndrome	DFNA2		 	 	 dominant	non-syndromic	form	of	hearing	loss	type	2	BFNS		 	 	 	 benign	familial	neotatal	seizures	PNH	 	 	 	 peripheral	nerve	hyperexcitability	Kv		 	 	 	 voltage-gated	potassium	(channel)	AED	 	 	 	 anti-epileptic	drug	RTG	 	 	 	 retigabine	ICA73	 	 	 	 ICA-06973				 			 ix		Acknowledgments		 Completing	 my	 Master’s	 thesis	 has	 been	 one	 of	 the	 most	 fulfilling	 and	challenging	experiences	I’ve	had.	Throughout	the	process	of	pursuing	my	degree,	I’d	like	to	thank	numerous	individuals	who	made	this	ride	that	much	better.	Firstly,	I’d	like	to	thank	my	supervisor,	Dr.	Harley	Kurata,	for	the	valuable	guidance	on	all	aspects	of	my	 project,	 the	 positive	mentorship	 throughout	 the	 degree,	 and	 for	 adding	 an	element	of	excitement	to	this	2-year	process	by	moving	to	Edmonton	halfway.	As	well,	thank	you	to	my	committee	members,	Dr.	Eric	Accili	and	Dr.	Filip	Van	Petegem	for	the	invaluable	advice	on	my	project.		I’d	also	like	to	thank	my	lab	members,	Victoria	Baronas,	Robin	Kim,	Caroline	Wang,	Michael	Yau,	and	Dr.	Runying	Yang	(specifically	for	molecular	biology	support),	for	 all	 the	 moral	 and	 technical	 support,	 and	 for	 an	 incredibly	 pleasant	 lab	environment.	 Thank	 you	 also	 to	 Wynne	 Leung	 and	 Jessica	 Yu	 for	 administrative	support	during	my	Master’s	degree.	Finally,	I’d	like	to	thank	my	family	and	friends	who	have	been	there	for	me	since	day	one	of	this	process.		 			 1	Chapter	1:	Introduction	and	Background	1.1	Kv	Overview		 Potassium	 channels	 are	 fundamental	 entities	 in	 physiology	 as	 K+	 ions	 are	essential	for	proper	functioning	of	the	heart,	kidneys,	muscles,	nerves,	and	digestive	system	(Pischalnikova	and	Sokolova,	2009).	These	channels	conduct	K+	ions	across	the	 membrane,	 along	 the	 electrochemical	 gradient	 for	 K+.	 A	 variety	 of	 cellular	processes	rely	on	appropriate	K+	channel	function,	including	maintenance	of	resting	membrane	potential,	regulation	of	cell	volume,	hormone	secretion,	and	modulation	of	 electrical	 impulses	 (Hille,	 2001;Pischalnikova	 and	Sokolova,	 2009).	There	 are	4	major	 families	 of	 potassium	 channels:	 voltage-gated	 (Kv),	 Ca2+-activated	 (Kca),	inward-rectifying	(Kir),	and	two-pore	(K2p),	with	Kv	being	the	largest	of	the	families,	encompassing	40	genes	and	further	divided	into	12	subfamilies	(Gutman	et	al.,	2005).	As	the	names	may	suggest,	the	families	differ	by	how	they	are	gated,	and	this	diversity	enables	channel	responses	to	a	variety	of	physiological	stimuli	(MacKinnon,	2003).		The	open	probability	(Po)	of	Kv	channels	depends	on	the	membrane	voltage,	with	channels	normally	closed	at	resting	voltages	around	-70	to	-90	mV	and	opened	upon	membrane	depolarization	(Sahoo	et	al.,	2014).	With	this	property,	Kv	channels	play	 significant	 roles	 in	 regulating	 neuronal	 and	 cardiac	 tissue	 excitability	 (Hille,	2001).	 However,	 they	 can	 also	 be	 found	 in	 neuroendocrine	 and	 endocrine	 cells,	skeletal	 muscle,	 placenta,	 lung,	 liver,	 and	 kidney	 tissues	 (Ju	 and	Wray,	 2002).	 In	excitable	 cells,	 Kv	 channels	 can	 counteract	 electrical	 excitation	 by	 stabilizing	 or	setting	the	resting	membrane	potential;	they	can	also	regulate	action	potential	shape			 2	or	frequency	(Sahoo	et	al.,	2014).	In	contrast,	Kv	channels	may	also	act	as	K+	transport	vehicles	in	non-excitable	cells	(Sahoo	et	al.,	2014).	It	is	not	surprising	that	with	their	wide	 range	 of	 roles	 that	mutations	 in	 Kv	 channels	 can	 result	 in	many	 severe	 and	inherited	 diseases	 such	 as	 episodic	 ataxia	 type	 1,	 deafness,	 epilepsy,	 or	 cardiac	arrhythmia	(Pischalnikova	and	Sokolova,	2009).	1.1.1	Overall	Structure	of	Kv	Channels		 Kv	channels	can	be	composed	of	α	and	β	subunits,	with	conduction	of	K+	across	the	lipid	bilayers	occurring	through	the	integral	membrane	α	subunit	(Biggin	et	al.,	2000).	 A	 functional	 Kv	 channel	 is	 tetrameric,	 with	 4	 identical	 or	 related	 subunits	(MacKinnon,	 1991;Li	 et	 al.,	 1992).	 While	 tetramerization	 is	 promoted	 by	 the	 N-terminus	 in	 Kv1-4	 channels,	 it	 is	 established	 by	 the	 C-terminus	 in	 Kv7	 and	 Kv11	channels	 (Biggin	 et	 al.,	 2000).	 Each	monomer	 contains	 6	 TM	 helices	 (S1-S6);	 the	central	pore	domain	is	made	up	of	S5	and	S6	from	all	4	subunits,	which	is	surrounded	by	4	VSD’s	composed	of	S1-S4	(Swartz,	2004;Tombola	et	al.,	2005).	A	small	portion	of	each	VSD	contacts	the	pore	domain	of	the	neighbouring	subunit	via	its	S4-S5	linker	that	runs	underneath	the	neighbouring	subunit	(Tombola	et	al.,	2005).		1.1.2	Ion	Selectivity	of	Kv	Channels	With	their	complex	yet	delicate	architecture,	Kv	channels	can	conduct	K+	ions	with	 1000-fold	 selectivity	 over	 Na+	 ions	 even	 though	 their	 atomic	 radii	 are	 not	significantly	different:	1.33Å	and	0.95Å,	respectively	(MacKinnon,	2003).	The	central	ion	conduction	pathway	is	encircled	by	the	4	subunits	that	make	up	the	pore,	with	each	subunit	contributing	two	transmembrane	α-helices	and	a	tilted	pore	helix	that	runs	half	way	through	the	membrane,	pointing	its	C-terminal	negative	‘end-charge’			 3	dipole	towards	the	ion	pathway	(Doyle	et	al.,	1998;Zhou	et	al.,	2001).	A	central	water-filled	cavity	sits	near	the	midpoint	of	the	membrane,	where	the	ion	pathway	is	almost	10Å	 in	 diameter	 (Doyle	 et	 al.,	 1998;Zhou	 et	 al.,	 2001).	 K+	 ions	 are	 thought	 to	 be	stabilized	at	the	centre	of	the	membrane	by	hydration	and	the	negative	end-charge	of	the	α-helices	directed	towards	the	pathway	(Roux	and	MacKinnon,	1999).	While	this	has	 been	 a	 generally	 accepted	 model,	 it	 has	 also	 been	 shown	 that	 substituting	positively	 charged	 residues	 at	 those	 central	 positions	 did	 not	 affect	 potassium	channel	function,	deeming	the	pore	helix	dipole	effect	to	be	less	prominent	(Chatelain	et	al.,	2005).		At	 the	 extracellular	 third	 of	 the	 ion	 pathway,	 K+	 ions	 are	 selected	 by	 the	selectivity	filter,	which	has	a	conserved	signature	sequence	of	GYG	or	GFG	(Doyle	et	al.,	 1998;Biggin	 et	 al.,	 2000;MacKinnon,	 2003).	 With	 4	 evenly	 spaced	 layers	 of	carbonyl	oxygen	atoms	and	a	single	layer	of	threonine	hydroxyl	oxygen	atoms,	4	K+	binding	 sites	 are	 formed	 (MacKinnon,	 2003).	 Since	 the	 arrangement	 is	 similar	 to	being	surrounded	by	water	molecules,	K+	 ions	can	be	dehydrated	and	bind	at	 low	energy	 cost,	with	 octahedral	 coordination	 by	 4	 oxygen	 atoms	 above,	 and	4	 below	(MacKinnon,	2003).	The	high	selectivity	of	K+	over	Na+	occurs	because	the	selectivity	structure	is	dependent	on	the	presence	of	K+;	conformational	change	induced	by	K+	allows	stronger	binding	(MacKinnon,	2003).	In	addition,	the	high	conduction	rate	of	K+	through	the	channel	is	due	to	repulsion	that	occurs	between	closely	spaced	K+	ions.	2	ions	are	present	in	the	filter	at	a	given	time;	as	an	ion	enters	from	one	side	of	the	filter,	repulsion	causes	another	to	exit	from	the	opposite	side	(MacKinnon,	2003).				 4	1.1.3	Kv	Channel	Gating	and	Voltage-Sensing	Like	K+	selectivity,	gating	works	similarly	in	all	potassium	channel	families.	In	response	 to	 a	 stimulus	 such	 as	 a	 change	 in	 membrane	 voltage	 for	 Kv	 channels,	conformational	 changes	 in	 the	 transmembrane	 voltage-sensing	 domain	 occur	 to	facilitate	 or	 trigger	 pore	 opening	 (MacKinnon,	 2003).	 The	 gate	 of	 the	 channel	 is	located	at	the	intracellular	end	of	the	pore	and	its	open	and	closed	conformations	are	controlled	by	a	conserved	glycine	gating	hinge;	the	inner	helices	can	obstruct	the	pore	in	the	closed	state	and	rotate	to	expand	the	intracellular	diameter	in	the	open	state	(MacKinnon,	2003;Grottesi	et	al.,	2005).	In	Kv	channels,	the	gate	is	linked	indirectly	to	the	voltage	sensor	by	the	α-helical	S4-S5	linker	that	contacts	the	S6	helix,	allowing	coupling	of	gate	conformation	to	voltage-sensor	movement	(Grottesi	et	al.,	2005).	As	the	channel	opens,	 charged	amino	acids	on	 the	S4	helix,	known	as	gating	charges,	move	through	the	membrane	electric	field	and	couple	electrical	work	to	opening	of	the	 channel	 pore	 (Schoppa	 et	 al.,	 1992;Sigworth,	 1994;Bezanilla,	 2000).	 The	 total	gating	 charge	 in	Shaker	 channels	 is	 estimated	 to	 be	 approximately	 14	 elementary	charges	(the	sum	of	the	charged	sidechains	and	the	net	fraction	of	the	transmembrane	voltage	difference	that	they	cross)	(Schoppa	et	al.,	1992;Aggarwal	and	MacKinnon,	1996;Seoh	et	al.,	1996).	It	has	been	demonstrated	that	the	first	4	arginine	residues	of	the	S4	helix	(located	every	third	position	on	the	helix),	contribute	most	significantly	to	this	gating	charge	(Tombola	et	al.,	2005).		 When	 the	 membrane	 depolarizes,	 the	 voltage	 sensor	 in	 each	 subunit	experiences	a	voltage-dependent	transition	from	a	resting	(R)	state	to	an	activated	(A)	state,	permitting	the	pore	to	open	(Bezanilla	et	al.,	1994;Zagotta	et	al.,	1994).	As			 5	all	4	subunits	transition	into	the	A	state,	the	pore	gate	opens	cooperatively	through	a	concerted	transition	which	is	weakly	voltage-dependent	(Ledwell	and	Aldrich,	1999).	While	 the	 full	mechanism	of	 the	 voltage-sensing	 and	 gating	 coupling	 is	 still	 being	investigated,	 it	 has	 been	 suggested	 that	 residues	 at	 the	 external	 side	 of	 the	 pore	domain	form	important	contacts	that	 ‘brace’	 the	voltage	sensor	domain	(Lee	et	al.,	2009b),	while	a	cluster	of	residues	formed	between	the	internal	side	of	the	S4	and	S5	helices	of	adjacent	subunits	are	crucial	for	the	concerted	opening	transition	(Soler-Llavina	et	al.,	2006).		1.2	KCNQ	Overview		 The	Kv7	family,	also	known	as	the	KCNQ	family,	has	5	known	members	(Kv7.1-7.5	or	KCNQ1-5);	 they	generally	 function	to	stabilize	a	negative	resting	membrane	potential	and	oppose	electrical	excitability	(Robbins,	2001;Delmas	and	Brown,	2005).	KCNQ	 channels	 have	 a	 canonical	 voltage-gated	 potassium	 channel	 structure,	 a	tetramer	with	 six	 transmembrane	domains	 in	 each	 subunit.	Unlike	many	other	Kv	families,	 Kv7	 subunits	 lack	 the	 N-terminal	 T1	 domain	 that	 is	 responsible	 for	tetramerization	 in	 Kv1-4	 (Xu	 et	 al.,	 1995;Long	 et	 al.,	 2005).	 Instead,	 Kv7	 subunits	possess	a	unique	tetramerization	domain	in	the	C-terminus	which	has	no	homology	with	 the	 T1	 domain	 (Schwake	 et	 al.,	 2006;Howard	 et	 al.,	 2007).	 In	 addition,	 Kv7	subunits	share	a	conserved	domain	in	the	proximal	C-terminal	region	near	S6,	which	contains	 positively	 charged	 residues	 that	 are	 thought	 to	 bind	 membrane	 lipid	phosphatidylinositol	4,5	bisphosphate	(PIP2)	(Delmas	and	Brown,	2005;Hernandez	et	al.,	2008b).		KCNQ	 channels	 are	 widely	 expressed	 in	 both	 neuronal	 and	 non-neuronal			 6	tissues	(Cooper,	2011).	While	KCNQ2-5	were	first	discovered	in	neurons	where	they	play	important	roles	in	neurotransmitter-stimulated	action	potential	firing,	KCNQ1	was	originally	recognized	for	its	role	in	cardiac	myocytes	that	contributes	to	cardiac	action	potential	 repolarization,	and	 is	not	believed	to	be	significantly	expressed	 in	neurons	(Jespersen	et	al.,	2005;Mackie	and	Byron,	2008).	It	has	now	been	shown	that	there	are	also	KCNQ	channels	in	vascular	smooth	muscle	cells	from	several	vascular	beds	where	they	assist	with	vascular	tone	regulation	(Mackie	and	Byron,	2008).		Functionally,	KCNQ2-5	subtypes	form	subunits	of	the	low-threshold	voltage-gated	potassium	channel	that	had	been	named	the	“M-channel”	(Adams	and	Brown,	1980;Brown,	1988).	They	activate	at	subthreshold	potentials,	starting	at	around	-60	mV,	do	not	 inactivate	 like	many	other	Kv	channels,	 and	generate	a	 steady	voltage-dependent	 outward	 current	 as	 a	 result	 (Brown	 and	 Passmore,	 2009).	 Moreover,	activation	 of	 these	 channels	 is	 relatively	 slow	 (Brown,	 1988).	 Neuronal	 KCNQ	channels	 can	 exert	 a	 significant	 dampening	 effect	 on	 repetitive	 burst-firing	 and	general	excitability	of	neurons	(Brown,	1988).	Not	surprisingly,	KCNQ2	and	KCNQ3	channels,	the	main	neuronal	players	in	the	family,	are	expressed	densely	in	the	axon	initial	segments	(AIS)	where	an	action	potential	is	initiated;	they	also	co-localize	with	sodium	channels	through	the	binding	to	the	cytoskeletal	protein	ankyrin	G	and	can	thus	 regulate	 the	 action	 potential	 threshold	 (Chung	 et	 al.,	 2006;Rasmussen	 et	 al.,	2007).	Through	multiple	 signaling	pathways,	neuronal	KCNQ	channels	can	control	somatic	excitability,	bursting,	and	neurotransmitter	release	throughout	the	nervous	system	(Hernandez	et	al.,	2008b).				 7	1.2.1	M-current		 KCNQ2-5	members	underlie	the	M-current.	The	name	of	this	slowly	activating	and	 deactivating	 potassium	 current	 is	 due	 to	 its	 suppression	 by	 stimulation	 of	muscarinic	 acetylcholine	 receptors	 now	 known	 to	 be	 coupled	 to	 PIP2	 hydrolysis	through	the	Gq	signaling	cascade	(Adams	and	Brown,	1980;Alfonso	et	al.,	1997;Wang	et	al.,	1998).	In	the	1960’s	and	1970’s,	it	was	shown	that	in	sympathetic,	cortical,	and	hippocampal	 neurons,	 muscarinic	 acetylcholine	 receptor	 agonists	 induced	 slow	membrane	 depolarization	 and	 decreased	 potassium	 conductance	 (Kobayashi	 and	Libet,	 1968;Weight	 and	 Votava,	 1970;Krnjevic	 et	 al.,	 1971;Kuba	 and	 Koketsu,	1976;Dodd	et	al.,	1981).	In	1980,	the	M-current	was	finally	identified	as	the	voltage-sensitive	K+	current	that	was	responsible	for	the	decreased	potassium	conductance	after	 muscarinic	 receptor	 stimulation	 (Adams	 and	 Brown,	 1980).	 Activated	 at	relatively	negative	potentials,	around	-60	mV,	close	to	the	resting	potential	for	many	excitable	 cells,	 the	M-current	 generates	 a	 resting	 outward	 current	 that	 influences	cellular	 electrical	 excitability	 (Mackie	 and	 Byron,	 2008).	 As	 well,	 this	 time-	 and	voltage-dependent	 K+	 current	 does	 not	 inactivate	 and	 can	 activate	 further	 upon	membrane	 depolarization,	 acting	 as	 a	 brake	 for	 neuronal	 firing	 (Hernandez	 et	 al.,	2008b;Maljevic	et	al.,	2010).	While	it	is	still	commonly	referred	to	as	the	M-current,	it	is	now	known	that	 the	current	 is	not	exclusively	regulated	by	muscarinic	receptor	activation,	but	also	by	activation	of	other	G-protein	coupled	receptors	(Mackie	and	Byron,	2008).	In	addition,	the	inhibition	of	the	M-current	appears	to	be	an	indirect	response	to	the	stimulation	of	these	receptors	(Marrion,	1997).	The	activation	of	Gq	receptors	leads	to	the	hydrolysis	of	PIP2	in	the	membrane	catalyzed	by	phospholipase			 8	(Marrion,	1997).	 	This	reduction	of	PIP2	levels	in	the	membrane	has	been	found	to	cause	closure	of	the	M-channel	(Brown	and	Passmore,	2009).	Furthermore,	PIP2	is	required	for	the	activation	of	the	channel,	with	its	major	binding	site	being	a	cluster	of	basic	amino	acids	in	the	C-terminus	of	KCNQ	channels	(Hernandez	et	al.,	2008b).	1.2.2	KCNQ	Channels	and	Disease	KCNQ1	The	 first	member	 of	 the	KCNQ	 family,	 KCNQ1,	was	 identified	 by	 positional	cloning	on	chromosome	11p15.5	in	families	with	Long	QT	syndrome	Type	I	(Wang	et	al.,	1996).	It	was	found	that	KCNQ1	α-subunits	co-assemble	with	KCNE	β-subunits	to	form	functional	channels	conducting	the	slow	cardiac	delayed	rectifier	K+	current,	IKs	(Barhanin	et	al.,	1996;Sanguinetti	et	al.,	1996).	In	the	heart,	IKs	plays	an	important	role	in	the	repolarization	of	the	cardiac	late-phase	action	potential	(Maljevic	et	al.,	2010).		Interestingly,	KCNQ1	is	the	only	member	of	the	family	that	is	unable	to	form	hetero-tetramers	with	other	KCNQ	subunits	(Maljevic	et	al.,	2010).	While	KCNQ1	can	form	 homomeric	 channels	 without	 the	 KCNE1	 β-subunits,	 giving	 rise	 to	 fast-activating	 potassium	 currents,	 co-assembly	 with	 KCNE1	 yields	 slow-activating	currents	with	increased	macroscopic	amplitudes	(Barhanin	et	al.,	1996;Sanguinetti	et	al.,	1996).	The	ratio	of	the	co-assembly	between	KCNE1	and	KCNQ1	is	still	an	ongoing	debate	(Nakajo	et	al.,	2010;Yu	et	al.,	2013;Plant	et	al.,	2014;Murray	et	al.,	2016).		Although	 KCNQ1	 may	 be	 most	 well-known	 for	 its	 activity	 in	 the	 heart,	KCNQ1/KCNE1	 channels	 are	 also	 expressed	 in	 various	 areas	 including	 the	 stria	vascularis	 of	 the	 inner	 ear,	 small	 intestine,	 pancreas,	 thyroid	 gland,	 forebrain	neuronal	networks,	brainstem	nuclei,	lungs,	GI	tract,	and	the	ovaries	(Jespersen	et	al.,			 9	2005;Goldman	et	al.,	2009).	In	the	inner	ear,	KCNQ1	is	thought	to	conduct	K+	current	into	the	scala	media,	where	K+-rich	endolymph	solution	is	generated	(Jespersen	et	al.,	2005).	In	the	small	intestine	tip	cells,	KCNQ1	was	found	to	regulate	sodium-coupled	glucose	uptake	(Jespersen	et	al.,	2005).	Moreover,	KCNQ1/KCNE1	channels	are	also	located	in	the	proximal	and	distal	tubules	of	the	nephron	and	seem	to	be	important	for	kidney	function	(Maljevic	et	al.,	2010).	Long	QT	Syndrome		 Long	 QT	 syndrome	 (LQTS)	 affects	 1/3000	 of	 the	 general	 population	 and	 is	characterized	by	an	increased	duration	of	the	QT	interval	in	the	electrocardiogram,	a	reflection	of	delayed	cardiac	repolarization	(Robbins,	2001;Zareba	and	Cygankiewicz,	2008).	 It	can	be	acquired	or	congenital,	and	sufferers	usually	show	few	symptoms	until	certain	events	such	as	strenuous	exercise,	stress,	or	drug	usage	(Viskin,	1999).	It	has	the	propensity	to	trigger	a	characteristic	re-entrant	arrhythmia	(torsades	de	pointes),	 which	 may	 lead	 to	 ventricular	 tachycardia	 that	 can	 self-terminate	 or	degenerate	 into	 lethal	ventricular	 fibrillation	(Robbins,	2001).	 	The	symptoms	can	include	palpitations,	 syncope,	 aborted	cardiac	arrest,	 and	sudden	death	or	 cardiac	arrest	(Zareba	and	Cygankiewicz,	2008).			 While	there	are	12	known	types	of	LQTS,	the	most	common	type	is	LQT1,	which	makes	up	about	50%	of	the	LQTS	patients	(Robbins,	2001;Zareba	and	Cygankiewicz,	2008).	Over	240	different	known	mutations	of	the	KCNQ1	gene	have	been	identified	in	patients	with	this	disease	(Lundby	et	al.,	2010).	An	autosomal	dominant	form	of	LQT1,	Romano-Ward	syndrome,	is	caused	by	the	presence	of	non-functional	mutant	channels	 that	 also	 inhibit	 the	 function	 of	 any	 wild	 type	 subunits	 by	 preventing			 10	assembly	(Chouabe	et	al.,	1997;Wollnik	et	al.,	1997).	As	well,	there	is	an	autosomal	recessive	 type	 of	 LQT1,	 Jervell	 and	 Lange-Nielsen	 syndrome	 (JLNS).	 In	 its	heterozygous	form,	there	is	weak	cardiac	dysfunction,	but	in	its	homozygous	form,	there	is	severe	cardiac	dysfunction	as	well	as	bilateral	deafness	(Robbins,	2001).	Short	QT	Syndrome		 At	the	other	end	of	the	spectrum,	patients	with	Short	QT	syndrome	(SQTS)	have	a	 constantly	 short	 QT	 interval	 on	 their	 ECG	 (Zareba	 and	 Cygankiewicz,	 2008).	Although	this	condition	is	far	rarer	than	LQTS,	patients	with	no	underlying	structural	heart	disease	can	experience	atrial	fibrillation,	syncopal	episodes,	as	well	as	sudden	cardiac	death	(Zareba	and	Cygankiewicz,	2008).	One	of	the	5	types	of	SQTS,	SQT2,	is	caused	by	gain-of-function	KCNQ1	mutations	that	increase	IKs	and	accelerate	cardiac	repolarization	(Zareba	and	Cygankiewicz,	2008).	KCNQ2	&	KCNQ3		 The	KCNQ2	and	KCNQ3	genes	were	discovered	using	two	approaches:	screening	of	a	human	brain	cDNA	library	with	a	KCNQ1-derived	sequence	and	positional	cloning	in	 families	 affected	 by	 benign	 familial	 neonatal	 seizures	 (BFNS)	 (Biervert	 et	 al.,	1998;Charlier	 et	 al.,	 1998;Singh	 et	 al.,	 1998;Yang	 et	 al.,	 1998).	 Both	 KCNQ2	 and	KCNQ3	subunits	have	been	found	in	most	regions	of	the	brain,	including	the	cortex,	cerebellum,	 basal	 ganglia,	 and	 hippocampus	 (Maljevic	 et	 al.,	 2010).	 While	 the	expression	pattern	of	these	channels	changes	during	development,	they	are	mainly	located	at	the	axon	initial	segments	(AIS)	(Devaux	et	al.,	2004;Maljevic	et	al.,	2008).		 KCNQ2	and	KCNQ3	channels	give	rise	to	non-inactivating	potassium	currents	that	 activate	 slowly	 upon	 depolarization	 (Maljevic	 et	 al.,	 2010).	 While	 KCNQ2			 11	homomeric	 channels	 can	 generate	 current,	 KCNQ3	 homemeric	 channels	 cannot.	Heteromeric	 KCNQ2/KCNQ3	 channels	 produce	 currents	 that	 are	 greater	 in	magnitude	than	the	homomeric	KCNQ2	channels,	with	possible	varying	expression	ratios	of	 the	 two	 subunits	 (Schroeder	 et	 al.,	 1998).	 It	 has	been	 shown	 that	 a	pore	alanine	 residue	 (315)	 in	 KCNQ3	 that	 is	 otherwise	 a	 threonine	 in	 the	 other	 KCNQ	channels,	 causes	KCNQ3	 to	 be	 retained	 in	 the	 endoplasmic	 reticulum,	 rather	 than	trafficked	to	the	cell	membrane	(Gomez-Posada	et	al.,	2010).	KCNQ3	with	the	A315T	mutation	can	form	homemeric	channels	and	generate	currents	that	are	very	large	in	magnitude	 (Gomez-Posada	 et	 al.,	 2010).	 These	 observations	 have	 led	 to	 the	understanding	that	KCNQ3	channels	are	intrinsically	unstable	and	therefore	depend	on	heteromerization	with	KCNQ2	for	function	(Gomez-Posada	et	al.,	2010).	Mutations	in	KCNQ2	and	KCNQ3	can	cause	BFNS,	peripheral	nerve	hyperexcitability	(PNH),	and	epileptic	encephalopathy	(Hart	et	al.,	2002;Maljevic	et	al.,	2010;Wang	et	al.,	2015).	BFNS		 BFNS	 is	 a	 rare	 autosomal	dominant	 epilepsy	 syndrome	with	a	penetrance	of	more	than	80%	(Maljevic	et	al.,	2010).	The	condition	starts	within	the	first	days	of	birth	with	frequent	and	often	clustered	partial	and	secondary	generalized	seizures	(Maljevic	et	al.,	2010).	While	these	indications	disappear	spontaneously	after	a	few	weeks	or	months,	there	is	about	a	15%	risk	of	recurring	seizures	later	in	life	for	these	patients	(Maljevic	et	al.,	2010).		 Although	there	are	more	than	30	known	BFNS	mutations,	the	majority	are	found	in	KCNQ2,	with	only	4	in	KCNQ3	(Coppola	et	al.,	2003;Neubauer	et	al.,	2008).	Various	types	 of	 mutations	 in	 KCNQ2	 including	 truncations,	 splice	 site	 defects,	 missense,			 12	nonsense,	or	frame-shift	mutations	have	been	found	to	cause	BFNS	(Lee	et	al.,	2009a).	Pore	mutations	do	not	seem	to	cause	a	dominant-negative	effect	on	wild-type	KCNQ2	channels,	but	KCNQ2	G271V	in	the	pore	has	been	found	to	cause	infantile	seizures	(Wang	et	al.,	2015).		Not	surprisingly,	BFNS	mutations	found	in	the	S4	of	KCNQ2	affect	channel	 gating	 and	 increase	 the	 threshold	 for	 channel	 activation	 (Maljevic	 et	 al.,	2010).	Mutations	found	in	the	C-terminus,	on	the	other	hand,	tend	to	impair	surface	expression	by	different	mechanisms	such	as	 reduced	protein	 stability	or	ability	 to	bind	calmodulin,	thereby	affecting	transport	to	the	membrane	surface	(Maljevic	et	al.,	2010).				 	Intriguingly,	seizures	in	BFNS	typically	only	occur	during	the	neonatal	period.	One	 possible	 explanation	 for	 this	 phenomenon	 is	 that	 expression	 of	 KCNQ2	 and	KCNQ3	may	increase	with	maturation	(Maljevic	et	al.,	2008).	In	addition,	there	may	be	a	developmental	 switch	 from	GABAergic	excitatory	 to	 inhibitory	actions,	which	may	 be	 vital	 for	 controlling	 transient	 generation	 of	 seizures;	 during	 the	 neonatal	period,	the	M-current	from	KCNQ2	and	KCNQ3	channels	may	be	the	only	available	inhibitory	 channels	 to	 act	 as	 a	 brake	 to	 neuronal	 excitability	 (Okada	 et	 al.,	2003;Safiulina	et	al.,	2008).	Peripheral	Nerve	Hyperexcitability	(PNH)		 Since	KCNQ2	channels	are	located	in	both	the	central	and	peripheral	nervous	systems,	mutations	can	also	result	in	PNH	(Maljevic	and	Lerche,	2014).	Patients	with	PNH	suffer	from	spontaneous	and	continuous	muscle	overactivity,	with	undulating	movements	of	the	distal	skeletal	muscle,	fasciculations,	cramps,	and	other	indications	generated	by	hyperexcitability	of	peripheral	motor	neurons	(Hart	et	al.,	2002).	So	far,			 13	only	2	mutations	in	KCNQ2	have	been	associated	with	PNH	and	they	are	both	found	at	arginine207	in	the	S4	voltage	sensor	(Maljevic	and	Lerche,	2014).	Epileptic	Encephalopathy		 Finally,	 mutations	 in	 KCNQ2	 have	 also	 been	 found	 to	 cause	 epileptic	encephalopathy	 with	 cognitive	 impairment.	 Patients	 present	 with	 a	 diversity	 of	syndromes	 characterized	 by	 early	 occurrence	 of	 seizures	 that	 are	 correlated	with	impaired	neurological	development;	they	have	pharmaco-resistant	neonatal	onset	of	seizures	with	a	strong	tonic	component	(Maljevic	and	Lerche,	2014).	Although	by	age	3,	 seizures	 typically	 stop,	 profound	 intellectual	 disability	 and	 motor	 impairment	persist	(Weckhuysen	et	al.,	2012;Milh	et	al.,	2013;Weckhuysen	et	al.,	2013).	For	this	more	severe	disease,	the	mutations	found	so	far	affect	functionally	crucial	parts	of	the	KCNQ2	channel:	 the	S4	voltage-sensor,	pore,	and	C-terminus	(Maljevic	and	Lerche,	2014).	Therefore,	it	is	evident	that	decreased	M-current	at	the	start	of	life	not	only	can	 cause	 seizures,	but	 also	affect	normal	neuromotor	development	 (Maljevic	 and	Lerche,	2014).	KCNQ4	&	KCNQ5		 KCNQ4	was	cloned	by	mapping	to	the	DFNA2	locus	for	a	form	of	nonsyndromic	dominant	deafness	(Kubisch	et	al.,	1999).	The	majority	of	KCNQ4	channels	have	been	found	in	outer	hair	cells	of	the	inner	ear,	and	specifically	the	Type	I	hair	cells	of	the	cochlea	(Kubisch	et	al.,	1999;Kharkovets	et	al.,	2000).	In	addition,	KCNQ4	is	located	in	the	vestibular	apparatus	and	a	number	of	nuclei	in	the	central	auditory	pathway	(Kharkovets	et	al.,	2000).	During	development,	the	change	in	expression	pattern	of	outer	hair	cells	is	in	parallel	with	the	onset	of	hearing,	alluding	to	the	potential	effect			 14	of	 KCNQ4	 on	 electrical	 properties	 of	 the	 outer	 hair	 cells	 as	 well	 as	 in	 lowering	intracellular	potassium	concentrations	(Maljevic	et	al.,	2010).		Last	but	not	least,	KCNQ5	has	been	cloned	(Schroeder	et	al.,	2000;Lerche	et	al.,	2000).	 However,	 not	 much	 is	 known	 about	 the	 channel	 other	 than	 that	 it	 forms	heteromeric	 channels	 with	 KCNQ2-4.	 Moreover,	 no	 disease	 has	 been	 found	 to	 be	associated	with	the	gene	so	far.	More	recently,	KCNQ	1,	4,	and	5	have	been	found	to	express	in	vascular	and	nonvascular	 smooth	muscles	 (Greenwood	 and	Ohya,	 2009;Stott	 et	 al.,	 2014).	 It	 is	speculated	that	these	channels	can	serve	as	potential	therapeutic	targets	for	diseases	such	as	irritable	bowel	syndrome,	constipation,	bladder	instability,	asthma,	and	pre-term	labour	due	to	their	role	in	regulating	nonvascular	smooth	muscle	activity	(Stott	et	al.,	2014).	DFNA2		 DFNA2	 is	 an	 autosomal	 dominant	 disease	 that	 causes	 hearing	 loss	 at	 high	frequencies	to	occur	in	patients’	twenties	and	thirties	and	can	progress	to	more	than	60	 dB	 with	 middle	 and	 low	 frequencies,	 within	 10	 years	 (Nie,	 2008).	 So	 far,	 8	missense	mutations	and	2	deletions	in	the	KCNQ4	gene	have	been	found	in	DFNA2	patients	with	diverse	clinical	phenotypes	(Nie,	2008).	While	there	are	still	much	to	be	known	about	this	channel	and	disease,	it	has	been	shown	that	patients	with	missense	mutations	tend	to	have	earlier	onset	of	and	all-frequency	hearing	loss	(Nie,	2008).	In	contrast,	patients	with	deletion	mutations	seem	to	have	later	onset	and	pure	high-frequency	hearing	loss	(Nie,	2008).	A	possible	explanation	of	the	relation	of	KCNQ4	malfunction	to	the	disease	is	that	loss	of	KCNQ4	currents	may	contribute	to	chronic			 15	K+	overload	in	the	outer	hair	cells,	leading	to	degeneration	and	progressive	hearing	loss	(Kharkovets	et	al.,	2006).	It	has	been	shown	that	KCNQ	channel	openers	such	as	retigabine	can	rescue	some	DFNA2	mutations,	shedding	light	on	potential	treatment	of	DFNA2	and	other	forms	of	deafness	involving	hair	cell	loss	(Leitner	et	al.,	2012).		1.3	PIP2	Regulation	of	KCNQ	1.3.1	PIP2		Since	ion	channels	are	expressed	in	the	cell	membrane,	it	is	important	to	note	the	 roles	 in	which	 phospholipids	 play	 in	 regulating	 channels.	 One	 specific	 type	 of	phospholipid	in	the	plasma	membrane,	phosphatidylinositol	4,5-bisphosphate	(PIP2),	has	been	known	to	bind	to	and	regulate	a	diversity	of	ion	channels	(Hilgemann	and	Ball,	1996;Hilgemann	et	al.,	2001;Suh	and	Hille,	2005;Suh	and	Hille,	2008).	An	anionic	lipid	 located	 in	 the	 inner	 leaflet	 of	 the	 surface	membrane,	 PIP2	makes	 up	 a	 small	fraction	(<1%)	of	 the	entire	pool	of	phospholipids	(McLaughlin	et	al.,	2002;Rusten	and	Stenmark,	2006).	It	is	synthesized	in	two	steps	from	phosphatidylinositol	(PI):	sequential	phosphorylation	steps	by	PI	4-kinase	and	PI(4)P5	kinase	at	 the	plasma	membrane	(Falkenburger	et	al.,	2010).	Importantly,	 PIP2	 acts	 as	 the	 principal	 substrate	 of	 phospholipases	 (PLC),	which	are	stimulated	by	over	50	hormone	receptors	that	couple	to	the	G	protein	Gq	and	 some	 receptor	 tyrosine	 kinases	 (Falkenburger	 et	 al.,	 2010).	 At	 the	 plasma	membrane,	PLC	cleaves	PIP2	into	two	potent	second	messengers,	lipid	diacylglycerol	and	 soluble	 inositol	 1,4,5-trisphosphate	 (Falkenburger	 et	 al.,	 2010).	 The	 latter	triggers	release	of	Ca2+	from	intracellular	stores,	which	leads	to	further	downstream	cascades	(Falkenburger	et	al.,	2010).				 16	1.3.2	PIP2	Effects	on	KCNQ	Channels		 Members	of	the	KCNQ	family	absolutely	require	PIP2	for	conducting	current,	as	demonstrated	 in	 a	 variety	 of	 experiments	 (Zaydman	 and	Cui,	 2014).	While	 it	was	previously	known	that	the	M-current	is	inhibited	by	muscarinic	receptor	activation,	it	is	now	believed	that	this	is	due	to	Gq-PLC	activation	leading	to	PIP2	hydrolysis	(Suh	and	Hille,	2002;Zhang	et	al.,	2003;Suh	et	al.,	2004).	Therefore,	it	is	not	surprising	that	PIP2	regulation	of	KCNQ	channels	is	physiologically	important	in	neurons	as	it	plays	a	role	in	neuroexcitability	(Delmas	and	Brown,	2005;Brown	et	al.,	2007).		In	addition,	mutations	in	KCNQ1	that	are	associated	with	Long	QT	syndrome	have	been	found	to	disturb	PIP2-dependent	activation	(Park	et	al.,	2005;Logothetis	et	al.,	2010;Zaydman	et	 al.,	 2013).	 Therefore,	 understanding	 how	PIP2	 interacts	with	 KCNQ	 channels	 is	essential	for	learning	further	about	channel	function	and	mechanisms.		1.3.3	Potential	PIP2	Binding	Sites		 In	 terms	 of	 PIP2	 structure,	 the	 lipid	 phosphate	 group	 appears	 to	 be	 key	 in	sustaining	current,	but	not	the	net	headgroup	charge,	the	acyl	chain	length,	nor	the	effects	of	lipids	on	membrane	curvature	(Schmidt	et	al.,	2006).	While	there	seems	to	be	a	shared	and	conserved	PIP2	binding	site	at	the	VSD-PGD	interface	(Zaydman	and	Cui,	2014),	PIP2	does	not	appear	to	directly	affect	VSD	activation	and	there	is	no	firm	evidence	 on	 whether	 or	 not	 PIP2	 influences	 the	 PGD	 opening	 in	 KCNQ	 channels	(Zaydman	and	Cui,	2014).	At	the	VSD-PGD	interface,	charge	neutralizing	and	charge	reversing	 mutations	 of	 basic	 residues	 decrease	 channel	 open	 probability	 while	simultaneously	 decreasing	 apparent	 affinity	 to	 exogenous	 PIP2	 (Hernandez	 et	 al.,	2008a).	Furthermore,	experiments	have	been	conducted	 in	KCNQ2	to	suggest	 that			 17	PIP2	preferentially	interacts	with	the	S4-S5	linker	during	the	open	state	and	with	the	S2-S3	loop	in	the	closed	state,	both	of	which	are	in	the	VSD-PGD	interface	(Zhang	et	al.,	2013).	Nevertheless,	much	has	yet	to	be	discovered	about	PIP2	and	its	interactions	with	KCNQ	channels;	in	the	meantime,	the	essential	role	of	PIP2	must	be	considered	when	studying	KCNQ	in	order	to	yield	meaningful	results	and	interpretations.		1.4	KCNQ	Channel	Openers	Epilepsy	 is	 one	 of	 the	 most	 common	 and	 serious	 chronic	 neurological	disorders,	and	affects	about	1%	of	 the	population	worldwide	(Sander,	2003).	This	disorder	is	diverse	in	presentation	and	can	be	generally	characterized	by	abnormal	synchronous	 and	 rhythmic	 neuronal	 activity	 causing	 repetitive	 epileptic	 seizures	(Orhan	et	al.,	2012).	Since	the	discovery	of	anticonvulsant	properties	of	phenobarbital	in	1912,	there	has	been	ongoing	development	of	antiepileptic	drugs	(AEDs),	although	roughly	30%	of	all	epileptic	patients	remain	resistant	to	current	pharmacotherapy	(Brodie	 and	 French,	 2000;Kwan	 and	 Brodie,	 2010).	 To	 date,	 several	 important	mechanisms	of	action	of	AEDs	have	been	uncovered,	including	blockage	of	voltage-gated	 sodium	 or	 calcium	 channels,	 enhancement	 of	 gamma-aminobutyric	 acid	(GABA)-mediated	 inhibitory	 neurotransmission,	 or	 attenuation	 of	 glutamate-mediated	excitatory	neurotransmission	(Perucca,	2005).		More	 recently,	 the	 fundamental	 role	 of	 KCNQ	 channels	 as	 regulators	 of	neuronal	 excitability	has	become	more	evident,	 given	 their	 ability	 to	maintain	 the	cell’s	resting	membrane	potential	and	reduce	sub-threshold	excitability	in	the	brain	(Gunthorpe	et	al.,	2012).	Therefore,	the	development	of	retigabine	(RTG)	as	a	first-in-class	potassium	channel	opener	AED	has	shed	light	on	new	approaches	for	treatment			 18	of	epilepsy.		1.4.1	Development	of	RTG		The	 structure	 of	 retigabine,	 or	 ezogabine	 (the	 US	 adopted	 name),	 was	designed	 based	 on	 two	 drugs	 known	 to	 modulate	 Kv7	 channels,	 linopirdine	 and	flupirtine	(Aiken	et	al.,	1995).	While	the	former	is	a	selective	Kv7	channel	blocker	and	exhibits	 pro-convulsive	 and	 cognitive	 enhancing	 effects,	 the	 latter	 is	 the	 first	 Kv7	channel	enhancer	used	as	an	analgesic	and	shows	anticonvulsant	properties	(Aiken	et	 al.,	 1995).	 Retigabine	 (RTG),	 chemically	 identified	 as	 ethyl	 N-[2-amino-4-4[(4-fluorophenyl)methylamino]pheny]	 carbamate,	 demonstrated	 the	 greatest	anticonvulsant	potency	among	several	 structural	analogues	of	 flupirtine	 that	were	synthesized	(Orhan	et	al.,	2012).	Developed	in	the	1980’s,	the	efficacy	of	RTG	shown	in	preclinical	models	supported	its	use,	especially	with	the	discovery	and	cloning	of	the	 new	 KCNQ	 family,	 found	 to	 be	 genetically	 linked	 to	 BFNS	 (Biervert	 et	 al.,	1998;Charlier	 et	 al.,	 1998;Singh	 et	 al.,	 1998).	 Following	 two	 large-scale	 Phase	 III	double-blind	placebo-controlled	trials	in	patients	with	partial	(focal)	epilepsy,	the	use	of	RTG	as	a	drug	and	KCNQ	channels	as	a	drug	 target	 for	epilepsy	were	validated	(Brodie	et	al.,	2010;French	et	al.,	2011).	Finally,	the	approval	of	RTG	by	both	the	US	FDA	and	European	Union	for	adjunctive	treatment	of	partial	onset	seizures	in	adults	arrived	after	more	than	20	years	of	preclinical	and	clinical	research	(Gunthorpe	et	al.,	2012).	However,	at	this	point,	RTG	is	not	yet	licensed	for	other	forms	of	epilepsy	or	diseases	(Orhan	et	al.,	2012).	Mechanism	of	RTG		RTG	 has	 been	 demonstrated	 through	 in	 vitro	 studies	 to	 activate	 Kv7.2/7.3			 19	channels	and	thereby	enhance	K+	conductance.	In	electrophysiological	experiments,	the	 most	 pronounced	 effect	 of	 RTG	 is	 a	 marked	 concentration-dependent	hyperpolarizing	 shift	 in	 the	 voltage-dependent	 activation	 curve	 of	 KCNQ	 channels	(Gunthorpe	 et	 al.,	 2012).	 This	 activation	 of	 the	 M-current	 results	 in	 membrane	hyperpolarization	towards	the	potassium	equilibrium	potential,	which	can	negatively	modulate	 neuronal	 firing	 rates	 and	 oppose	 neuronal	 hyperexcitability	 as	 seen	 in	epileptic	 seizures	 (Orhan	 et	 al.,	 2012).	 While	 RTG	 can	 increase	 K+	 current	 by	increasing	the	open	probability	of	KCNQ	channels,	 it	does	not	seem	to	alter	single-channel	 conductance	 of	 individual	 KCNQ2/3	 channels	 (Gunthorpe	 et	 al.,	 2012).	Instead,	RTG	increases	the	rate	of	channel	activation	and	decelerates	channel	closure	(Main	et	al.,	2000;Wickenden	et	al.,	2000;Tatulian	et	al.,	2001).	It	is	important	to	note	that	RTG	can	activate	Kv7.2-7.5	channels	but	not	the	Kv7.1	cardiac	isoform	at	human	therapeutic	serum	levels,	which	reduces	the	risk	for	potential	cardiac	adverse	effects	(Orhan	et	al.,	2012).			 Through	many	studies,	the	molecular	mechanism	of	RTG	has	been	elucidated.	A	conserved	Trp	residue	in	the	S5	of	Kv7.2-7.5	members	has	been	found	to	be	required	for	RTG’s	action.	A	recent	study	established	that	the	effect	relies	on	a	molecule	of	RTG	forming	an	H-bond	with	this	pore	region	residue	(Kim	et	al.,	2015).	Side	Effects	of	RTG	While	 RTG	 is	 the	 first	 and	 best-characterized	 Kv7	 channel	 opener	 at	 the	moment,	serving	as	an	important	chemical	tool	to	study	the	therapeutic	potential	of	Kv7	 channel	 modulation,	 it	 indeed	 has	 room	 for	 improvement	 (Tatulian	 et	 al.,	2001;Tatulian	and	Brown,	2003).	RTG	potentiates	channels	other	than	KCNQs,	such			 20	as	GABA-receptors,	 and	has	 little	 subtype	 specificity	 between	different	Kv7	 family	members	(Gribkoff,	2003;van	Rijn	and	Willems-van,	2003).	Thus,	various	retigabine	side	effects	have	arisen,	including	undesired	effects	in	the	CNS	and	in	smooth	muscle.	For	 example,	 dizziness,	 somnolence,	 fatigue,	 hallucinations,	 confusion,	 and	 speech	disorders	have	been	reported	in	certain	patients	(Orhan	et	al.,	2012).	As	well,	negative	effects	 on	 the	urological	 system	 leading	 to	urinary	 retention	has	been	a	 recurring	problem	 in	 patients	 (Streng	 et	 al.,	 2004).	 It	 has	 also	 been	 reported	 that	 RTG	 can	induce	 blue-grey	 mucocutaneous	 discoloration	 of	 the	 skin,	 nail,	 oral	 mucous	membrane,	conjunctiva,	and	retina	(Garin	et	al.,	2014).	Therefore,	various	new	Kv7	channel	openers	have	been	synthesized	and	hopefully	new	compounds	will	address	shortcomings	attributed	to	RTG.		1.4.2	Other	KCNQ	Openers		Besides	RTG,	other	compounds	have	been	identified	with	potentiating	activity	on	 Kv7	 channels.	 Since	 the	 development	 of	 RTG,	 a	 number	 of	 pharmaceutical	companies	have	used	the	structure	of	RTG	as	a	template	for	constructing	new	and	improved	Kv7	openers	(Castle,	2010).	Several	classes	of	compounds	such	as	oxindole	analogues	(eg.	BMS-204352)	are	potent	Kv7.2-7.5	activators	while	others	like	R-L3	activate	 Kv7.1	 (Xiong	 et	 al.,	 2008).	 As	 well,	 fenamates	 including	 diclofenac,	meclofenamic	acid,	NH6,	and	NH29,	some	widely	used	NSAIDs	that	non-selectively	inhibit	COX-1	and	COX-2,	are	relatively	potent	Kv7.2/7.3	activators	(Munster	et	al.,	2002).	Zinc	pyrithione	(ZnPy),	used	for	controlling	dandruff	and	treating	psoriasis,	has	also	been	shown	to	be	a	strong	Kv7	activator,	potentiating	all	Kv7	channels	except	Kv7.3	 (Xiong	 et	 al.,	 2007).	 Icagen	 has	 synthesized	 various	 pyridinyl	 benzamide			 21	compounds	 such	 as	 ICA-27243,	 ICA-110381,	 and	 ICA-069673,	 which	 appear	 to	selectively	open	certain	Kv7	channels	over	others	(Castle,	2010).	New	Potential	Mechanisms	of	KCNQ	Openers		While	it	is	validated	that	RTG	acts	through	the	Trp	residue	in	the	pore	region	of	KCNQ2-5,	some	of	the	new	KCNQ	channel	openers	appear	to	act	through	a	different	mechanism	and	binding	site.	This	is	suggested	as	various	compounds	including	ZnPy,	NH29,	 and	 the	 Icagen	 compounds	 still	 activate	 the	Trp	 to	 Leu/Phe	mutant	 that	 is	insensitive	to	RTG	(Xiong	et	al.,	2007;Peretz	et	al.,	2010;Boehlen	et	al.,	2013;Padilla	et	 al.,	 2009).	 Furthermore,	 a	 number	 of	 these	 compounds	 such	 as	 NH29	 and	 the	Icagen	compounds	have	been	suggested	to	interact	with	the	VSD	rather	than	the	pore	region	of	KCNQ	(Padilla	et	al.,	2009;Peretz	et	al.,	2010).	1.5	Overview	of	Chapter	3:	Sequence	Determinants	of	Subtype-specific	Actions	of	KCNQ	Channel	Openers			 In	this	study,	an	extensive	analysis	of	the	compound	ICA-06973	(referred	to	as	ICA73	from	here	onwards)	has	been	done	to	determine	its	mechanism	of	action	that	selectively	 activates	 KCNQ2	 channels	 but	 not	 KCNQ3	 channels.	 Through	 the	construction	of	chimeric	channels	between	KCNQ2	and	KCNQ3	as	a	starting	point,	we	noticed	that	ICA73	interacted	with	the	VSD	of	KCNQ2	(in	particular,	the	S3-S4	region)	for	its	effect,	rather	than	with	the	pore	region	as	RTG	does.	Using	a	non-radioactive	rubidium	efflux	assay	as	a	screening	tool	and	whole-cell	patch	clamping,	we	sifted	through	numerous	mutants	in	the	S3-S4	to	pinpoint	the	key	residues	that	allow	ICA73	to	 produce	 its	 substantial	 effect	 on	 KCNQ2	 but	 not	 KCNQ3.	 We	 identified	 two	mutations	in	the	S3,	F168L	and	A181P,	that	significantly	reduced	ICA73	effect.	While			 22	the	 F168L	 mutation	 completely	 abolished	 the	 ICA73	 effect,	 the	 A181P	 mutation	exhibited	a	surprising	effect	where	the	channel	loses	most	of	the	hyperpolarizing	shift	by	ICA73	but	not	the	current	potentiation.	In	addition,	we	found	that	RTG	could	still	activate	these	two	mutants,	suggesting	two	different	mechanisms	of	action	for	RTG	and	 ICA73.	 This	 hypothesis	 is	 confirmed	 as	 KCNQ2[W236F],	 a	 mutant	 that	 is	insensitive	to	RTG,	can	be	fully	activated	by	ICA73.		1.6.	Overview	of	Chapter	4:	In-depth	Analysis	of	Voltage	Sensor	Residues	Involved	in	ICA73	Effects	on	KCNQ2			 While	Chapter	3	focused	on	S3-S4	residues	in	KCNQ2	and	KCNQ3	required	for	ICA73’s	 dramatic	 effect	 on	 KCNQ2,	 the	 first	 half	 of	 the	 VSD	 (S1-S2)	 may	 also	 be	involved	in	the	effect	as	seen	in	the	rubidium	efflux	assay.	Since	the	S1-S2	sequence	of	 KCNQ2	 and	 KCNQ3	 differ	 sizably,	 Q2	 +	 Q3[S1]	 and	 Q2	 +	 Q3[S2]	 chimeras,	 in	addition	to	point	and	cluster	mutations	were	made	to	 tease	out	potential	residues	that	 may	 contribute	 to	 the	 ICA73	 effect.	 Patch	 clamp	 experiments	 did	 not	 find	particular	residues	that	were	specific	to	reducing	ICA73	effect.			 To	 further	 decipher	 how	 ICA73	 may	 be	 binding	 to	 KCNQ2,	 molecular	simulations	were	carried	out	to	look	at	solvent	accessible	pockets	that	could	make	up	the	 potential	 binding	 site.	 Electrophysiology	 indicated	 that	 mutating	 E130,	 an	important	residue	in	voltage-gated	potassium	rchannels	that	acts	as	a	voltage	sensor	counter	 charge,	 ablates	 the	 ICA73	 hyperpolarizing	 shift,	 but	 not	 the	 current	potentiation	 effect.	 This	 observation	 has	 not	 been	 noted	 in	 previous	 studies.	Numerous	 details	 in	 this	 study	 as	well	 as	 the	 previous	 chapter	 have	 opened	 new	insight	 on	 KCNQ	 channel	 openers	 and	 their	 heterogeneity	 as	 well	 as	 how	 slight			 23	differences	in	KCNQ2-5	structures	can	allow	drugs	to	target	specific	subtypes.	This	can	 aid	 in	 future	 drug	 design	 which	 may	 eliminate	 non-specific	 targets	 and	 thus	reduce	side	effects.												 24	Chapter	2:	Materials	and	Methods	2.1	KCNQ2	and	KCNQ3	Channel	Constructs	Mutant	 channels	 were	 derived	 from	 human	 KCNQ2	 or	 KCNQ3	 genes	(originally	in	pTLN	vector-	gifts	from	Dr.	Taglialatela	and	Dr.	T.	Jentsh),	expressed	in	pcDNA3.1	 (-)	 plasmid	 (Invitrogen,	 Carlsbad,	 CA).	 KCNQ3*	 channels	 refer	 to	KCNQ3[A315T],	 carrying	 a	 point	 mutation	 that	 allows	 homomeric	 expression	 of	KCNQ3	 (Gomez-Posada	 et	 al.,	 2010).	 Chimeras	 between	 KCNQ2	 and	 KCNQ3	were	constructed	 using	 an	 overlapping	 PCR	 method.	 Flanking	 primers	 were	 used	 to	amplify	respective	segments	of	KCNQ2	and	KCNQ3.	PCR	approaches	were	then	used	to	sequentially	combine	overlapping	 fragments	until	all	necessary	segments	of	 the	chimera	were	 incorporated.	The	break	points	 for	 the	 chimeras	 generated	were	 as	follows.	For	Q2+Q3[S1-S2]	channels,	KCNQ2	residues	89-148	were	substituted	with	KCNQ3	 residues	 117-178.	 	 For	 Q2+Q3[S3-S4]	 channels,	 KCNQ2	 residues	 153-207	were	substituted	with	KCNQ3	residues	183-236.	For	Q2+Q3[S5-S6]	channels,	KCNQ2	residues	239-324	were	substituted	with	KCNQ3	residues	268-363.	 	For	Q2+Q3[S1]	channels,	KCNQ2	residues	89-115	were	substituted	with	KCNQ3	residues	117-145.	For	 Q2+Q3[S2]	 channels,	 KCNQ2	 residues	 115-149	were	 substituted	with	 KCNQ3	residues	 145-178.	 Point	 mutants	 in	 KCNQ2	 were	 constructed	 using	 a	 2-step	overlapping	PCR	method.	All	constructs	were	subcloned	into	pcDNA3.1(-)	using	NheI	and	 EcoRI	 restriction	 enzymes	 and	 verified	 by	 Sanger	 sequencing	 approaches	(Genewiz	or	University	of	Alberta	Applied	Genomics	Core).				 25	2.2	Cell	Culture	and	Whole-cell	Patch	Clamp	Recordings	HEK293	 cells	 were	 cultured	 in	 50	 mL	 polystyrene	 tissue	 culture	 flasks	(Falcon)	 in	 DMEM	 (Invitrogen)	 supplemented	 with	 10%	 FBS	 and	 1%	 penicillin-streptomycin.	Cells	were	grown	in	an	incubator	at	5%	CO2	and	37	°C.	Cells	were	plated	into	6-well	plates	and	co-transfected	with	plasmids	encoding	the	channel	of	interest	and	GFP	using	jetPRIME	DNA	transfection	reagent	(Polyplus	Transfection).	After	24	hours	 of	 incubation	 with	 transfection	 reagent,	 cells	 were	 split	 onto	 sterile	 glass	coverslips	 and	 electrophysiological	 experiments	 were	 conducted	 1	 day	 later.	 Whole-cell	 patch	 clamp	 recordings	 were	 performed	 using	 extracellular	solution	consisting	of	135	mM	NaCl,	5	mM	KCl,	2.8mM	NaAcetate,	1	mM	CaCl2(2H2O),	1	mM	MgCl2(6H2O),	 and	10	mM	HEPES,	with	pH	adjusted	 to	7.4,	 and	 intracellular	solution	containing	135	mM	KCl,	5mM	EGTA,	and	10	mM	HEPES,	with	pH	adjusted	to	7.3.	Retigabine	and	ICA069073	were	stored	as	100	mM	stocks	 in	DMSO,	and	were	diluted	to	working	concentrations	in	extracellular	solution	on	each	experimental	day.		Glass	pipette	tips	were	manufactured	using	soda	lime	glass	(Fisher	Scientific)	and	had	open	tip	resistances	of	1-3	MΩ	using	the	standard	experimental	solutions.	Recordings	were	 filtered	 at	 5	 kHz	 and	 sampled	 at	 10	 kHz	 using	 a	Digidata	 1440A	 (Molecular	Devices)	 controlled	by	 the	pClamp	10	 software	 (Molecular	Devices).	Experimental	compounds	were	purchased	from	Toronto	Research	Chemicals	(retigabine)	or	Tocris	(ICA-069673).		2.3	Two-electrode	Voltage	Clamp	Recordings	Complementary	 RNA	was	 transcribed	 from	 the	 cDNA	 of	 several	 constructs	using	the	mMessage	mMachine	Kit	(Ambion).	Stage	V–VI	Xenopus	laevis	oocytes	were			 26	prepared	 as	 previously	 described	 and	 were	 injected	 with	 cRNA.	We	 used	 female	Xenopus	laevis	frogs	100	g	or	greater	in	size	and	the	oocytes	were	prepared	using	a	protocol	approved	by	the	University	of	British	Columbia	Animal	Care	Committee,	in	accordance	 with	 the	 Canadian	 Council	 for	 Animal	 Care	 guidelines.	 Oocytes	 were	incubated	post	injection	for	12–96	h	at	18	°C	before	recording.	We	recorded	voltage-clamped	potassium	currents	in	standard	Ringers	solution	(in	mM):	116	NaCl,	2	KCl,	1	MgCl2,	 0.5	 CaCl2,	 5	 HEPES	 (pH	 7.4)	 using	 an	 OC-725C	 voltage	 clamp	 (Warner,	Hamden,	CT).	Glass	microelectrodes	were	backfilled	with	3	M	KCl	and	had	resistances	of	0.1–1	MΩ.	Data	were	 filtered	at	5	kHz	and	digitized	at	10	kHz	using	a	Digidata	1440A	 (Molecular	 Devices)	 controlled	 by	 the	 pClamp	 10	 software	 (Molecular	Devices).		2.4	Non-radioactive	Rb+	Efflux	Assay	HEK293	cells	were	plated	into	24-well	plates	and	co-transfected	with	channel	of	interest,	IRK1,	and	GFP	protein,	using	jetPRIME	DNA	transfection	reagent	(Polyplus	Transfection).	 Cells	were	 incubated	 in	 the	presence	of	 transfection	 reagent	 for	 24	hours	and	another	day	in	the	presence	of	regular	media.	2	days	post	transfection,	cells	were	 loaded	with	Rb+	 loading	media	 (1mM	RbCl	 in	DMEM	with	10%	FBS	and	1%	penicillin-streptomycin)	for	2	hours.	This	was	followed	by	washing	twice	with	0	K+	buffer	 (140mM	NaCl,	1.2	mM	MgSO4,	2.5mM	CaCl2,	10mM	HEPES	at	pH	7.4).	Cells	were	then	incubated	in	600	μL	0	K+	buffer,	100	K+	activating	buffer	(40	mM	NaCl,	100	mM	 KCl,	 1.2	mM	MgSO4,	 2.5	 CaCl2,	 and	 10	mM	HEPES	 at	 pH	 7.4),	 or	 0	 K+	 buffer	supplemented	with	respective	concentrations	of	ICA73.	200	μL	of	assay	aliquots	were	removed	at	multiple	time	points	(5,	10,	and	20	minutes)	and	cells	were	lysed	with	1%			 27	SDS	RIPA	lysis	buffer	following	completion	of	assay.	Rb+	concentration	in	supernatant	was	 measured	 by	 flame	 atomic	 absorption	 spectroscopy	 using	 Aurora	 Biomed	ICR8000	instrument.	Amount	of	Rb+	efflux	was	calculated	as	a	fraction	of	total	Rb+	loaded	(sum	of	Rb+	remaining	in	lysed	cells	and	Rb+	extruded	from	cells).	Normalized	Rb+	efflux	in	drug	solution	as	presented	in	data	is	calculated	by	(Effluxwith	drug	–	Effluxin	0	K+)/(Effluxin	100	K+	-	Effluxin	0	K+).	Only	the	20-minute	time	point	data	(mean	±	s.e.m.)	are	presented	for	simplicity.		2.5	Data	Analysis	Voltage	dependence	of	 channel	 activation	was	 fitted	with	a	 standard	 single	component	Boltzmann	equation	of	the	form	G/Gmax=1/(1+e−(V−V1/2)/k)	where	V1/2	is	the	voltage	where	channels	exhibit	half-maximal	activation,	and	k	 is	a	slope	 factor	reflecting	 the	 voltage	 range	 over	which	 an	 e-fold	 change	 in	 Po	 is	 observed.	 Note:	channels	that	exhibit	a	dramatic	response	to	ICA73	were	not	fitted.									 			 28	Chapter	3:	Sequence	Determinants	of	Subtype-specific	Actions	of	KCNQ	Channel	Openers	3.1	Introduction	Retigabine	(RTG)	is	a	recently	approved	anti-epileptic	drug	currently	used	as	an	 adjunct	 add-on	 treatment	 for	pharmaco-resistant	 epilepsy	 (Miceli	 et	 al.,	 2008).	Retigabine	 and	 its	 closely	 related	 analog	 flupirtine	 are	 currently	 the	 only	 voltage-gated	 potassium	 channel	 openers	 approved	 for	 human	 use.	 Their	 mechanism	 of	action	is	well-described	at	the	molecular	level	–	they	are	thought	to	interact	with	a	Trp	sidechain	located	in	the	pore-forming	S5	segment	of	KCNQ2-5	channel	subunits,	and	cause	a	pronounced	hyperpolarizing	shift	in	the	voltage-dependence	of	channel	activation	(Schenzer	et	al.,	2005;Lange	et	al.,	2009).	Importantly,	RTG	exhibits	little	specificity	between	KCNQ2-5	channel	 subunits,	 and	off-target	effects	 in	peripheral	tissues	including	the	bladder	have	been	a	recurring	problem	in	patients	(Martyn-St	et	al.,	2012;Brickel	et	al.,	2012).	However,	RTG	has	served	as	an	important	chemical	tool	to	study	the	therapeutic	potential	of	KCNQ	channel	modulation,	and	as	a	template	for	the	development	of	 further	analogs	that	may	exhibit	better	subunit	selectivity	and	centrally-restricted	actions	(Xiong	et	al.,	2008).		Following	the	recognition	of	the	therapeutic	benefit	of	retigabine,	a	variety	of	compounds	 have	 been	 identified	 that	 act	 as	 KCNQ	 channel	 openers	 (Miceli	 et	 al.,	2011).		Several	classes	of	compounds	such	as	oxindole	analogues	(eg.	BMS-204352)	are	potent	KCNQ2-5	activators	while	others	like	R-L3	activate	KCNQ1	(Xiong	et	al.,	2007).	As	well,	fenamates	including	diclofenac	and	meclofenamic	acid,	widely	used			 29	NSAIDs	 that	 non-selectively	 inhibit	 COX-1	 and	 COX-2,	 are	 also	 relatively	 effective	activators	 of	KCNQ2	 and	KCNQ2/3	heteromers	 (Munster	 et	 al.,	 2002;Peretz	 et	 al.,	2007;Peretz	et	al.,	2010).	Zinc	pyrithione	(ZnPy),	used	for	controlling	dandruff	and	treating	 psoriasis,	 has	 also	 been	 shown	 to	 be	 a	 strong	 KCNQ	 activator,	 causing	potentiation	of	all	KCNQ	channel	currents	except	KCNQ3	(Xiong	et	al.,	2007).	Some	commercially	 available	 compounds	 have	 been	 suggested	 to	 exhibit	 some	 subtype	selectivity,	 including	 ML-213,	 and	 a	 family	 of	 pyridinyl	 benzamide	 compounds	including	ICA-27243,	ICA-110381,	and	ICA-069673	(Wickenden	et	al.,	2008;Gao	et	al.,	2010;Blom	et	al.,	2010).		With	 this	 progress	 in	 discovery	 of	 KCNQ	 openers,	 fundamental	 principles	underlying	 their	 actions	 are	 beginning	 to	 be	 revealed.	 For	 example,	 we	 recently	demonstrated	the	requirement	for	a	hydrogen	bond	donor	in	the	KCNQ3	Trp265	(S5	Trp)	 side	 chain	 that	 is	 thought	 to	 interact	with	 retigabine	 (Kim	et	 al.,	 2015).	 This	chemical	property	correlated	with	the	polarity	of	a	hydrogen	bond	acceptor	carbonyl	found	in	many	KCNQ	channel	openers.	Drugs	such	as	ICA-069673,	with	very	weak	polarity	at	this	position	exhibit	very	weak	effects	on	KCNQ3	channels.	Also,	a	close	structural	analog	with	much	stronger	bond	polarity	(ML-213)	exhibited	much	more	potent	activation	of	KCNQ3	(Yu	et	al.,	2011).	Given	this	understanding	of	the	chemical	basis	of	drug	action	at	the	putative	retigabine	binding	site,	we	sought	to	understand	reports	of	 the	activating	effects	of	 ICA-069673	(and	other	close	analogs)	on	KCNQ	channels.	Several	reports	suggested	the	possibility	that	certain	KCNQ	activators	may	act	 on	 a	 distinct	 site	 present	 in	 certain	 KCNQ	 subtypes.	 For	 example,	 a	 family	 of	diclofenac	derivatives	were	shown	to	be	insensitive	to	mutation	of	the	S5	Trp	residue			 30	that	is	essential	for	retigabine	actions,	and	may	interact	with	the	VSD	rather	than	the	pore	region	of	KCNQ	(Padilla	et	al.,	2009;Peretz	et	al.,	2010).	In	 this	 study,	 we	 have	 exploited	 the	 reported	 subunit	 specificity	 of	 some	retigabine	analogs	 to	better	define	 the	mechanism	of	action	of	 these	drugs,	and	to	identify	sequence	determinants	that	underlie	subunit	specificity.	Our	study	identifies	two	 nearby	 residues	 in	 the	 voltage-sensing	 domain	 of	 KCNQ2	 that	 are	 unique	 to	ICA73-sensitive	channels,	are	required	for	ICA73	actions,	and	do	not	markedly	alter	channel	gating	or	retigabine	sensitivity.	These	residues	demonstrate	unambiguously	that	KCNQ	channel	openers	can	act	independently	of	the	retigabine	binding	site	with	a	distinct	mechanism.	Our	study	also	highlights	multi-pronged	effects	of	ICA73,	which	causes	 a	 marked	 hyperpolarizing	 shift	 of	 KCNQ2,	 and	 a	 dramatic	 potentiation	 of	current.	These	two	effects	can	be	separated	by	point	mutations	in	the	KCNQ2	voltage-sensing	domain.	3.2	Results	3.2.1	Subtype-specific	Effects	of	ICA069673		 Retigabine	 is	 a	 non-selective	 activator	 of	 KCNQ2-5	 channels,	 with	 a	 well-defined	binding	site	in	the	pore	domain	(Lange	et	al.,	2009;Kim	et	al.,	2015).	Certain	KCNQ	activators,	 including	ML-213,	 ICA	069673	 (referred	 to	 as	 ICA73	 and	 ICA81,	respectively,	 from	here	onwards),	 and	Zn-pyrithione,	 have	been	 reported	 to	 show	some	subtype	specificity,	although	some	reports	are	conflicting	and	the	determinants	of	 this	 specificity	 remain	 unclear	 (Xiong	 et	 al.,	 2007;Boehlen	 et	 al.,	 2013).	 For	example,	we	recently	observed	that	ML-213,	described	as	a	KCNQ2-specific	activator,	could	 also	 activate	 KCNQ3*	 (KCNQ3[A315T],	 see	Methods)	 channels	 with	 greater			 31	potency	than	retigabine	(Yu	et	al.,	2010;Kim	et	al.,	2015).	We	continued	to	investigate	the	effects	of	other	known	KCNQ	openers	on	KCNQ2	and	KCNQ3*	channels	(Fig.	3-1A).				 	Figure	3-1	ICA069673	exhibits	subtype-specificity	for	KCNQ2	over	KCNQ3	channels.	(A)	Chemical	structures	of	retigabine	and	ICA069673	(‘ICA73’).	(B,C)	Conductance-voltage	relationships	for	KCNQ2	and	KCNQ3*	were	generated	using	recordings	from	Xenopus	 laevis	 oocytes	 expressing	 each	 respective	 channel.	 KCNQ2	 parameters	 of	activation	were	(control	V1/2	=	-37	±	2	mV,	k=	10	±	1	mV;	retigabine	V1/2	=	-61	±	1	mV,	k	=	17	±	1	mV,	n	=	5).	KCNQ3	parameters	of	activation	were	(control	V1/2	=	-43	±	1	mV,	k=	8	±	1	mV;	ICA73	V1/2	=	-50	±	2	mV,	k	=	11	±	2	mV;	retigabine	V1/2	=	-103	±	1	mV,	k	=	7	±	1	mV,	n	=	5).				 We	 recognized	 that	 ICA069673	 (‘ICA73’)	 and	 retigabine	 have	 dramatically	different	 effects	 on	 KCNQ2	 vs.	 KCNQ3*	 channels	 (Fig.	 3-1B,C).	 Consistent	 with	previous	reports,	retigabine	caused	a	leftward	shift	of	the	V1/2	of	activation	in	both	KCNQ2	and	KCNQ3*	(Schenzer	et	al.,	2005).	While	the	effects	of	retigabine	are	more	pronounced	 in	 KCNQ3*,	 these	 findings	 reflect	 that	 retigabine	 is	 a	 relatively	 non-selective	 KCNQ	 channel	 activator.	 In	 contrast,	 30	 µM	 ICA73	 induced	 a	 dramatic	hyperpolarizing	shift	of	KCNQ2	channel	activation,	while	even	higher	concentrations			 32	(100	µM)	elicited	relatively	negligible	effects	on	KCNQ3*	(Fig.	3-1C).	Based	on	these	effects	of	ICA73,	we	set	out	to	 investigate	specific	differences	between	KCNQ2	and	KCNQ3*	 that	 underlie	 subtype-specific	 drug	 effects.	 It	 is	 noteworthy	 that	 some	previous	studies	have	highlighted	an	alternative	binding	site	in	the	voltage-sensing	domain	 of	 KCNQ	 channels	 for	 certain	KCNQ	openers.	However,	 thus	 far,	 potential	residues	 identified	 that	 influence	 drug	 effects	 tend	 to	 be	 highly	 conserved	 and	essential	 for	 normal	 activation	 gating	 of	 Kv	 channels	 (e.g.	 KCNQ2	 positions	 E130,	F137,	 and	 S4	 residues	L197,	R198,	 and	R201),	 and	do	not	 provide	 a	 rationale	 for	understanding	 subtype	 specific	 effects	 (Tao	 et	 al.,	 2010;Pless	 et	 al.,	 2011;Li	 et	 al.,	2013b;Brueggemann	et	al.,	2014).		3.2.2	Potentiation	and	Shift	in	Voltage-dependent	Gating	of	KCNQ2	It	 should	 also	 be	 noted	 that	 ICA73	 exhibits	 two	 clear	 effects	 on	 KCNQ2	channels:	a	shift	in	the	voltage-dependence	of	activation,	and	a	potentiation	of	current	at	voltages	where	channel	activation	is	saturated.		These	effects	are	often	not	obvious	in	 published	work	 due	 to	 a	 common	 practice	 of	 normalizing	 conductance-voltage	relationships.	These	multiple	effects	of	 ICA73	on	KCNQ2	are	apparent	 in	exemplar	traces	(Fig.	3-2A,B),	and	in	conductance-voltage	relationships	normalized	to	the	peak	conductance	in	control	conditions	(Fig.	3-2C).	These	data	highlight	both	the	shift	in	voltage-dependence	 of	 activation,	 and	 the	 approximate	 doubling	 of	 peak	 channel	current	by	ICA73	even	at	voltages	that	saturate	the	conductance-voltage	relationship.	The	maximal	ICA73-mediated	gating	shift	 is	much	larger	than	retigabine	-	so	 large	that	 channels	 cannot	be	 closed	completely	even	with	very	negative	voltage	pulses	(illustrated	by	sample	tail	currents	at	-130	mV,	Fig.	3-2D).				 33		Figure	 3-2	 ICA73	 potentiates	 KCNQ2	 currents	 and	 induces	 a	 large	hyperpolarizing	shift	of	the	conductance-voltage	relationship.	(A,B)	Exemplar	patch	clamp	current	traces	of	WT	KCNQ2	(expressed	in	HEK	cells)	in	control	conditions	or	30	µM	ICA73.	Cells	were	held	at	-80	mV,	and	pulsed	between	-150	mV	and	+20	mV	for	2	s,	with	tail	currents	measured	at	-20	mV.	(C)	Conductance-voltage	relationships	for	KCNQ2	wild	type,	normalized	to	peak	conductance	in	control	for	each	cell	(mean	±	s.e.m)	(control	V1/2	=	-40	±	5	mV,	k=	12	±	1	mV;	n	=	4).	(D)	Exemplar	sweeps	to	elicit	tail	currents	in	WT	KCNQ2	wild	type	pulsed	to	-130	mV	in	control	or	ICA73	(from	a	holding	voltage	of	+20	mV).		3.2.3	ICA73	Acts	within	the	VSD	We	investigated	the	channel	elements	responsible	for	ICA73-selective	effects	on	KCNQ2	over	KCNQ3,	using	a	chimeric	approach	of	substituting	transmembrane	domains	in	KCNQ2	(ICA73-sensitive)	with	corresponding	(ICA73-insensitive)	KCNQ3	sequence	 (Fig.	 3-3A-C).	 Whole-cell	 patch	 clamp	 experiments	 revealed	 that	substitution	 of	 the	 KCNQ3	 pore	 (S5-S6)	 into	 KCNQ2	 largely	 preserved	 ICA73			 34	sensitivity,	with	persistence	of	both	a	large	hyperpolarizing	gating	shift	and	current	potentiation	 by	 ICA73	 (Fig.	 3-3F,I).	 In	 contrast,	 substitution	 of	 the	 KCNQ3	 S3-S4	sequence	into	KCNQ2	rendered	channels	completely	insensitive	to	ICA73,	abolishing	both	 the	 gating	 shift	 and	 current	 potentiation	 effects	 (Fig.	 3-3E,H).	 Finally,	substitution	of	the	KCNQ3	S1-S2	domain	caused	a	reduced	ICA73	effect	that	appeared	to	preserve	current	potentiation,	but	abolish	most	or	all	of	the	drug-mediated	shift	of	activation	gating	(Fig.	3-3D,G).	These	results	together	highlight	the	voltage-sensing	domain	(S1-S4)	as	a	likely	site	for	ICA73	subtype	selectivity	(Padilla	et	al.,	2009),	with	a	particular	importance	of	the	S3-S4	segments.	These	findings	also	highlight	that	the	gating	 shift	 and	 current	 potentiation	 effects	 may	 be	 observed	 separately,	 as	demonstrated	by	the	Q2+Q3[S1,S2]	chimera	(Fig.	3-3A,D,G)		 35						Figure	3-3	Substitution	of	KCNQ3	VSD	into	KCNQ2	alters	channel	sensitivity	to	ICA73.		(A-C)	 Cartoon	 representations	 of	 chimeric	 channels	 of	 KCNQ2	 (grey)	 and	 KCNQ3	(red).	(D-F)	Conductance-voltage	relationships	for	(D)	Q2+	Q3[S1,S2]	(control	V1/2	=	-9	±	1	mV,	k=	8	±	1	mV;	ICA73	V1/2	=	-22	mV	±	5	mV,	k	=	5	±	1	mV,	n	=	5),	(E)	Q2	+	Q3[S3-S4]	(control	V1/2	=	-40	±	3	mV,	k=	11	±	1	mV;	ICA73	V1/2	=	-39	±	2	mV,	k	=	10	±	1	mV,	n	=	4),	and	(F)	Q2+	Q3[S5-S6]	(control	V1/2	=	-25	±	7	mV,	k=	11	±	2	mV;	n	=	5)	normalized	to	peak	conductance	in	control.	(G-I)	Sample	current	traces	for	G)	Q2+	Q3[S1,S2],	(H)	Q2	+	Q3[S3-S4],	and	(I)	Q2+	Q3[S5-S6]	illustrating	the	lack	of	ICA73	effect	 when	 S3-S4	 of	 KCNQ3	 is	 substituted	 into	 KCNQ2.	 In	 all	 panels,	 error	 bars	represent	s.e.m.		 			 36	3.2.4	Identification	of	Amino	Acid	Determinants	of	ICA73	Subtype	Specificity		In	order	to	identify	residues	in	the	KCNQ2	voltage-sensing	domain	that	confer	ICA73	specificity,	we	used	sequence	alignments	of	KCNQ2	and	KCNQ3	(Fig.	3-4A)	to	guide	 mutagenesis	 of	 KCNQ2	 (to	 corresponding	 KCNQ3	 residues)	 at	 positions	throughout	S3	and	S4.	In	addition,	due	to	a	large	number	of	residue	differences	in	S1	and	 S2,	 we	 generated	 two	 additional	 chimeras	with	 either	 S1	 or	 S2	 from	 KCNQ3	substituted	into	KCNQ2.	We	employed	a	non-radioactive	Rb+	efflux	assay	to	screen	these	mutants	for	ICA73	sensitivity	(Li	et	al.,	2013a).	Since	ICA73	effectively	activates	KCNQ2	channels	even	at	very	hyperpolarized	potentials	(Fig.	3-2C),	ICA73-mediated	Rb+	 efflux	 in	 hyperpolarizing	 solutions	 was	 used	 to	 identify	 mutants	 that	 retain	ICA73-sensitivity.	Using	this	approach,	we	identified	the	KCNQ2	F168L	and	KCNQ2	A181P	mutations	 as	 highly	 perturbative	 to	 ICA73	 sensitivity	 (Fig.	 3-4B).	 It	 is	 also	noteworthy	 that	 the	 S1	 chimera	 and	 S2	 chimera	 both	 retained	 significant	 ICA73	sensitivity	 (in	 contrast	 to	 the	 S1-S2	 chimera	 described	 in	 Fig.	 3-3).	 This	 finding	suggested	to	us	that	there	may	be	important	interactions	between	S1	and	S2	that	play	a	 role	 in	 ICA73	 sensitivity	 (Fig.	 3-3D,G),	 but	 that	 swapping	 individual	KCNQ2	and	KCNQ3	residues	may	not	be	a	powerful	approach	to	 identify	these	differences.	We	have	focused	the	remainder	of	our	study	on	the	S3-S4	residues	identified	in	this	initial	scan,	 as	 these	 are	 previously	 unrecognized	 determinants	 of	 subtype	 specificity	 of	KCNQ	activators.					 37		Figure	3-4	Rubidium	efflux	 screen	 identifies	 two	S3	mutations	 that	diminish	ICA73	response.	(A)	Alignment	of	KCNQ2-5	sequences.	Differences	between	KCNQ2	and	KCNQ3	were	used	to	identify	candidate	non-conserved	residues	in	the	VSD	that	may	be	essential	for	ICA73	sensitivity.		(B)	A	rubidium	efflux	assay	was	used	to	scan	ICA73	responses	from	mutants	as	indicated.	Normalized	ICA73	response	was	calculated	as	the	ratio	of	the	response	in	ICA73	(in	hyperpolarizing	solution),	to	the	maximal	rubidium	efflux	observed	 in	 depolarizing	 solution	 (see	 Materials	 and	 Methods).	 (C)	 Structural	representation	 of	 a	 molecular	 model	 of	 a	 single	 KCNQ2	 subunit	 highlighting	 all	candidate	residues	screened	(red),	F168L	and	A181P	(blue),	and	Trp236	(yellow).					 38	3.2.5	Aromatic	Residues	at	KCNQ2	Residue	168	Preserve	ICA73	Sensitivity	KCNQ2[F168L]	 channels	 generated	 functional	 currents	 with	 activation	kinetics	 and	 voltage-dependence	 similar	 to	wild-type	 KCNQ2	 (Fig.	 3-5).	 However,	ICA73	had	virtually	no	effect	on	 these	channels	 (Fig.	3-5A-C),	consistent	with	data	from	Rb+	efflux	experiments.	The	F168L	mutation	abolished	both	the	ICA73-mediated	hyperpolarizing	 gating	 shift,	 and	 current	 potentiation	 (Fig.	 3-5B).	 Also,	 ICA73	 had	virtually	 no	 effect	 on	 kinetics	 of	 channel	 closure	 (Fig.	 3-5C).	We	 tested	 additional	aromatic	 substitutions	 at	 position	 168	 (Fig.	 3-5D,E).	 Conductance-voltage	relationships	 suggest	 that	 the	 aromatic	 substitutions	 preserve	 potentiation	 and	gating	 effects	 of	 ICA73,	 although	 the	 magnitude	 of	 ICA73	 effects	 was	 not	 as	pronounced	in	the	F168Y	mutant	(this	mutant	also	caused	an	intrinsic	gating	shift	in	the	absence	of	drug,	Fig.	3-5D,E).	This	essential	residue	is	located	in	an	interesting	region	 at	 the	 cytoplasmic	 side	 of	 the	 S3	 transmembrane	 helix	 (Fig.	 3-5F).	 In	previously	published	models	of	 the	KCNQ2	open	 state	 (Miceli	 et	 al.,	 2011;Gourgy-Hacohen	et	 al.,	 2014),	 F168	 is	 in	 close	proximity	with	 a	 conserved	 lysine	 (KCNQ2	K219)	in	the	S4-S5	linker,	and	a	conserved	arginine	(KCNQ2	R213)	at	the	cytoplasmic	side	of	the	S4	segment.	It	is	also	possible	that	F168	could	come	into	close	contact	with	other	S4	charged	sidechains	in	alternative	voltage	sensor	configurations.					 39			Figure	3-5	Functional	characterization	of	KCNQ2[F168L]	mutant	channels.		(A)	Exemplar	patch	clamp	current	recordings	of	KCNQ2[F168L].	Cells	were	held	at	-80	mV,	and	pulsed	between	-140	mV	and	+40	mV	for	2	s,	with	tail	currents	measured	at	 -20	 mV.	 (B)	 Conductance-voltage	 relationships	 of	 KCNQ2[F168L].	 The	conductance-voltage	relationship	in	ICA73	is	normalized	to	the	peak	conductance	in	control	conditions	for	each	individual	cell	(data	are	mean	±	s.e.m.)	(control	V1/2	=	-13	±	2	mV,	k=	9	±	1	mV;	ICA73	V1/2	=	-10	±	7	mV,	k	=	15	±	4	mV,	n	=	4).	(C)	Time	constants	 (τ)	of	 channel	 closure	were	measured	by	pulsing	 to	 a	 range	of	negative	voltages	 from	 a	 holding	 potential	 of	 +20	 mV;	 inset,	 sample	 tail	 currents	 of	KCNQ2[F168L]	 at	 -130	 mV.	 (D,E)	 Conductance-voltage	 relationships	 of	KCNQ2[F168Y]	(control	V1/2	=	 -7	±	5	mV,	k=	19	±	3,	n	=	4)	 	and	KCNQ2[F168W]	(control	 V1/2	 =	 -29	 ±	 9	mV,	 k=	 17	 ±	 2	mV,	 n	 =	 3),	 collected	 as	 in	 panel	 (A).	 (F)	Structural	 model	 of	 the	 KCNQ2	 open	 state,	 depicted	 as	 a	 ‘bottom	 view’	 of	 the	intracellular	side	of	the	channel	transmembrane	domain.	KCNQ2	residue	F168	is	in	close	 proximity	 with	 R213	 and	 K219,	 suggesting	 possible	 interactions	 that	 may	underlie	the	role	of	F168.						 40	3.2.6	Proline	at	Position	181	in	KCNQ2	Disrupts	ICA73	Effects	Surprisingly,	 with	 more	 detailed	 characterization,	 we	 observed	 that	 the	KCNQ2[A181P]	 retained	 pronounced	 potentiation	 of	 peak	 current,	 but	 a	 much	smaller	shift	of	voltage-dependent	activation	(Fig.	3-6A,B).	This	behavior	was	similar	to	the	Q2+Q3[S1,S2]	chimera	(Fig.	3-3A,D,G),	illustrating	once	again	that	the	gating	shift	and	current	potentiation	effects	do	not	necessarily	coincide.	Tail	current	kinetics	also	 illustrate	 channel	 susceptibility	 to	 the	 drug,	 although	 ICA73-mediated	deceleration	of	channel	closure	is	not	as	dramatic	as	observed	in	WT	KCNQ2	(Fig.	3-6C,	Fig.	3-2D).	Since	prolines	can	break	alpha-helical	structures,	we	tested	additional	substitutions	of	either	a	bulky	residue	(Leu,	high	helical	propensity),	or	a	small	and	flexible	 residue	 (Gly,	 low	 helical	 propensity,	 but	 still	 amenable	 to	 alpha-helical	configuration),	at	position	181.	ICA73	had	a	significant	effect	on	both	mutants	(Fig.	3-6D),	 suggesting	 that	 A181	 may	 not	 be	 essential	 for	 drug	 binding	 or	 a	 helical	configuration	of	S3,	but	the	proline	present	in	KCNQ3	may	deform	the	top	of	the	S3	helix	(Fig.	3-6F)	and	alter	the	channel	response	to	ICA73.			 		 			 41			Figure	 3-6	 Functional	 characterization	 of	 KCNQ2[A181P]	 mutant	 channels	illustrates	the	separable	nature	of	current	potentiation	and	gating	shift.		(A)	Exemplar	patch	clamp	current	recordings	of	KCNQ2[A181P].	(B)	Conductance-voltage	 relationships	 of	 KCNQ2[A181P].	 The	 conductance-voltage	 relationship	 in	ICA73	is	normalized	to	the	peak	conductance	in	control	conditions	for	each	individual	cell	(data	are	mean	±	s.e.m.)	(control	V1/2	=	-20	±	2	mV,	k=	11	±	1	mV;	ICA73	V1/2	=	-45	±	3	mV,	k	=	2	±	2	mV,	n	=	5).	 (C)	Time	constants	 (τ)	of	 channel	 closure	were	measured	by	pulsing	to	a	range	of	negative	voltages	from	a	holding	potential	of	+20	mV;	 inset,	 sample	 tail	 currents	 of	 KCNQ2[A181P]	 at	 -130	mV.	 (D,E)	 Conductance-voltage	relationships	of	KCNQ2[A181G]	(control	V1/2	=	-10	±	6	mV,	k=	11	±	2	mV,	n	=	3)	and	KCNQ2[A181L]	(control	V1/2	=	-18	±	8	mV,	k=	12	±	1	mV,	n	=	3),	collected	as	in	panel	(A).	(F)	Molecular	model	of	the	voltage-sensing	domain	of	KCNQ2	channels	in	the	open	state.	Residue	A181	is	located	on	the	extracellular	side	of	the	S3	segment.				 42	3.2.7	ICA73-insensitive	Mutations	Preserve	Retigabine	Sensitivity		Experiments	thus	far	suggest	that	ICA73	and	retigabine	likely	act	via	different	binding	sites	and	different	mechanisms.	However,	it	should	be	noted	that	there	have	been	no	direct	binding	studies	of	KCNQ	activators	to	these	channels,	and	 it	can	be	challenging	 to	 distinguish	 drug	 binding	 from	 other	 effects	 on	 channel	 gating	 or	mechanisms	that	couple	drug	binding	to	the	channel	gate.	To	further	investigate	the	effects	of	the	A181P	and	F168L	mutations,	and	the	effects	of	retigabine	versus	ICA73,	we	 compared	 the	 effects	 of	 these	 drugs	 on	 various	mutations	 known	 to	 influence	KCNQ	 activators.	 KCNQ2[W236F]	 mutant	 channels	 have	 disrupted	 retigabine	sensitivity,	 but	 retain	 sensitivity	 to	 ICA73	 (Fig.	 3-7A).	 KCNQ2[A181P]	 or	 [F168L]	mutant	channels,	shown	here	to	exhibit	disrupted	ICA73	sensitivity,	retain	sensitivity	to	 retigabine	 (Fig.	3-7B,C).	Taken	 together	with	 the	 relatively	 innocuous	effects	of	these	mutations	on	channel	gating	in	the	absence	of	activator	compounds,	these	data	suggest	that	ICA73	and	retigabine	act	on	different	channel	sites,	and	that	the	A181P	and	F168L	are	not	intrinsically	disruptive	of	channel	structure	or	activation	gating	(since	 channels	 gate	 relatively	 normally,	 and	 retain	 sensitivity	 to	 the	 established	channel	activator	retigabine).									 43			Figure	3-7	ICA73-insensitive	mutants	do	not	perturb	retigabine	sensitivity.	(A-C)	 Conductance	 voltage	 relationships	 for	 (A)	 retigabine-insensitive	 mutant	KCNQ2[W236F]	(control	V1/2	=	-45	±	1	mV,	k=	8	±	1	mV;	retigabine	V1/2	=	-47	±	2	mV,	 k	 =	 8	 ±	 1	 mV,	 n	 =	 5),	 and	 the	 ICA73-insensitive	 mutants	 (B)KCNQ2[F168L]	(control	V1/2	=	-30	±	3	mV,	k=	10	±	2	mV;	retigabine	V1/2	=	-53	±	5	mV,	k	=	14	±	2	mV,	 n	 =	 5)	 and	 (C)]KCNQ2[A181P]	 (control	 V1/2	 =	 -45	 ±	 4	 mV,	 k=	 12	 ±	 2	 mV;	retigabine	V1/2	=	-71	±	8	mV,	k	=	10	±	2	mV,	n	=	4),	in	control	conditions	or	100	µM	retigabine	 as	 indicated.	 Note	 that	 the	 KCNQ2[F168L]	 and	 [A181P]	 mutations	exhibited	variable	channel	rundown	in	the	presence	of	retigabine	(no	potentiation	as	observed	 with	 ICA73),	 so	 their	 conductance-voltage	 relationships	 have	 been	normalized	to	1	for	clarity.		3.2.8	ICA73-sensitivity	can	be	Transferred	to	KCNQ3		 We	investigated	whether	the	presence	of	KCNQ2	S3	residues	F168	and	A181	can	introduce	ICA73	sensitivity	into	KCNQ3.	We	substituted	the	residues	back	into	KCNQ3	 at	 corresponding	 positions	 L198F	 and	 P211A.	While	we	were	 not	 able	 to	express	 the	 KCNQ3[L198F]	 alone,	 we	 detected	 functional	 currents	 from	KCNQ3[P211A]	as	well	as	KCNQ3[L198F/P211A]	channels.	Both	channels	behaved	similarly	as	wild	type	KCNQ3	in	the	absence	of	ICA73,	yielding	large	non-inactivating	currents	 in	 control	 conditions	 (Fig.	 3-8A,B).	 Interestingly,	 both	 constructs	 also	responded	to	ICA73	much	more	strongly	than	wild	type	KCNQ3,	as	the	conductance-voltage	relationships	were	shifted	by	about	25-30	mV.	This	effect	is	milder	than	what	is	observed	with	KCNQ2	channels,	and	no	consistent	current	potentiation	by	ICA73			 44	was	 observed.	 Taken	 together,	 these	 results	 propose	 that	 there	 are	 other	 factors	involved	 in	 the	 ICA73	effect	but	 that	 the	 residues	we	 identified	are	 indeed	crucial	players.		Figure	3-8	 ICA73	effect	 can	be	partially	 rescued	by	 substitution	of	F168	and	A181	into	KCNQ3.	(A-C)	Conductance-voltage	relationship	of	(A)	KCNQ3*	(control	V1/2	=	-52	±	4	mV,	k	=	6	±	2	mV,	n	=	5;	100	µM	ICA73	V1/2	=	-61	±3	mV,	k	=	8	±2	mV,	n	=	5),	(B)	KCNQ3*	P211A	(control	V1/2	=	-53	±	3	mV,	k	=	5	±	1	mV,	n	=	4;	100	µM	ICA73	V1/2	=	-77	±	6	mV,	k	=	8	±	1	mV,	n	=	4),	and	(C)	KCNQ3*	L198F/P211A	(control	V1/2	=	-54	±	3	mV,	k	=	7	±	1	mV,	n	=	5;	100	µM	ICA73	V1/2	=	-80	±	3	mV,	k	=	12	±	2	mV,	n	=	5)	normalized	to	peak	conductance	in	given	condition.	In	all	panels,	error	bars	represent	s.e.m.		3.3	Discussion	The	development	of	retigabine	and	flupirtine	as	 ‘first-in-class’	voltage-gated	potassium	channel	openers	has	led	to	efforts	to	further	develop	and	understand	the	pharmacophore	 of	 this	 emerging	 drug	 class	 (Bentzen	 et	 al.,	 2006;Xiong	 et	 al.,	2008;Gao	et	al.,	2010;Peretz	et	al.,	2010;Blom	et	al.,	2010;Kim	et	al.,	2015).	Retigabine	exhibits	 little	 subtype	 specificity	 between	 KCNQ2-5,	 so	 a	 more	 detailed	understanding	of	the	mechanism	of	action	of	KCNQ	openers	may	contribute	to	the	development	of	more	specific	or	potent	analogs.	It	is	now	recognized	that	the	lack	of	subtype	specificity	of	retigabine	arises	because	of	its	essential	interaction	with	a	S5	Trp	residue	that	is	conserved	in	retigabine-sensitive	KCNQ2-5	subunits	(Lange	et	al.,			 45	2009).	Several	KCNQ	openers	have	been	reported	to	exhibit	some	subtype	selectivity	but	the	mechanism	and	sequence	determinants	underlying	this	specificity	have	been	unclear	(Padilla	et	al.,	2009;Yu	et	al.,	2011;Brueggemann	et	al.,	2014).	In	addition,	the	effects	of	KCNQ	openers	are	often	studied	in	heteromeric	KCNQ	channel	mixtures,	so	it	is	unclear	whether	individual	KCNQ	subtypes	may	differentially	contribute	to	drug	sensitivity.		In	this	study,	we	exploited	dramatic	differences	in	ICA73	effects	in	KCNQ2	vs.	KCNQ3	to	identify	specific	residues	that	are	essential	for	ICA73	subtype	specificity.	ICA73	 induces	 a	 very	 large	 hyperpolarizing	 shift	 of	 the	 KCNQ2	 (but	 not	 KCNQ3)	conductance-voltage	 relationship,	 together	 with	 a	 large	 potentiation	 of	 current	magnitude	–	both	effects	far	exceed	what	is	typically	reported	for	retigabine.	Using	a	chimeric	 approach,	 we	 identified	 S3	 residues	 A181	 and	 F168	 in	 KCNQ2	 that	 are	essential	for	normal	ICA73	sensitivity	(Figs.	4-6).	Interestingly,	our	findings	highlight	that	 certain	 mutations	 (KCNQ2[A181P]	 or	 the	 Q2+Q3[S1,S2]	 chimera)	 dissociate	gating	shift	effects	from	effects	on	current	potentiation.	Previous	 investigations	 have	 reported	 voltage	 sensor-mediated	 effects	 of	certain	KCNQ	openers	(Padilla	et	al.,	2009;Peretz	et	al.,	2010;Gao	et	al.,	2010).	For	example,	 a	 class	 of	 diclofenac-derived	 compounds	 was	 reported	 to	 interact	 with	conserved	voltage-sensing	residues	in	KCNQ2	(Peretz	et	al.,	2010).	Also,	a	chimeric	study	 suggested	 that	 the	 voltage	 sensor	 plays	 an	 important	 role	 in	mediating	 the	effects	 of	 ICA73	 and	other	 related	 compounds	 (Padilla	 et	 al.,	 2009).	 Lastly,	 recent	work	has	demonstrated	that	mutation	of	KCNQ2	residues	E130	and	F137	influence	sensitivity	 to	 ICA73	 and	 other	 closely	 related	 compounds	 (Li	 et	 al.,			 46	2013b;Brueggemann	 et	 al.,	 2014).	 However,	 these	 residues	 are	 highly	 conserved	residues	among	voltage-gated	potassium	channels,	serving	important	roles	as	voltage	sensor	 counter	 charges	 (E130)	 or	 as	 the	 ‘gating	 charge	 transfer	 center’	 (F137)	(Papazian	et	al.,	1995;Tao	et	al.,	2010;Pless	et	al.,	2011;Pless	et	al.,	2014).	Mutations	at	 these	 positions	 often	 have	 significant	 effects	 on	 channel	 function,	 and	 cannot	account	for	the	ICA73	subtype	specificity	for	KCNQ2	over	KCNQ3	(since	both	channels	have	identical	residues	at	these	positions).					Our	 study	 identifies	 KCNQ2	 F168	 as	 an	 essential	 determinant	 of	 ICA73	sensitivity.	KCNQ2[F168L]	mutant	channels	were	completely	unresponsive	to	ICA73,	exhibiting	no	hyperpolarizing	shift	or	current	potentiation	in	the	presence	of	the	drug	(Fig.	5).	Based	on	our	findings	thus	far,	an	aromatic	side	chain	at	this	position	appears	to	 be	 required	 for	 ICA73	 sensitivity.	 In	 molecular	 models	 of	 KCNQ2,	 the	 F168L	position	is	located	at	a	convergence	of	many	positively	charged	sidechains	including	a	 voltage-sensing	 arginine	 (R213),	 and	 a	 lysine	 (K219)	 in	 the	 S4-S5	 linker.	 The	requirement	 for	 an	 aromatic	 sidechain	 may	 involve	 this	 proximity	 to	 positively	charged	 side	 chains,	 or	 may	 be	 related	 to	 positioning	 of	 S3	 near	 the	 membrane	interface.		In	any	case,	both	Lys219	and	Arg213	are	almost	certainly	conformationally	mobile	during	channel	gating	and	we	are	pursuing	a	more	detailed	understanding	of	the	interactions	between	these	residues	with	F168	in	different	channel	states.		The	 KCNQ2	 A181P	 mutation	 had	 a	 very	 unexpected	 outcome	 on	 ICA73	response:	 the	 drug-mediated	 hyperpolarizing	 gating	 shift	 was	 virtually	 abolished,	although	 the	 current	 potentiation	 effect	 was	 preserved	 (Fig.	 6).	 This	 observation	highlights	 the	 importance	 of	 monitoring	 both	 of	 these	 distinct	 parameters	 when			 47	characterizing	KCNQ	openers	–	as	noted	earlier,	conductance-voltage	relationships	are	often	normalized,	and	this	data	transformation	may	mask	the	current	potentiating	effect	of	ICA73	or	other	openers	(particularly	when	it	occurs	together	with	a	 large	gating	shift).	Since	the	current	potentiation	effect	of	ICA73	persists	in	KCNQ2[A181P],	we	speculate	that	this	mutation	does	not	abolish	drug	binding,	but	rather	alters	how	drug	 interactions	 influence	 the	 gating	 process.	 Supporting	 this	 suggestion	 is	 that	ICA73	 interaction	 clearly	 decelerates	 channel	 closure,	 despite	 not	 significantly	changing	the	voltage-dependence	of	channel	opening.	We	also	observed	that	ICA73	sensitivity	was	preserved	with	a	variety	of	substitutions	at	position	A181,	suggesting	that	the	native	alanine	may	not	directly	contribute	to	binding.	Rather,	the	presence	of	a	proline	(as	in	KCNQ3)	may	alter	the	nature	of	the	response	to	the	drug,	rather	than	altering	 binding.	 Importantly,	 there	 may	 be	 additional	 residues/mechanisms	generating	subtype	specificity,	as	KCNQ5	has	also	been	reported	to	be	insensitive	to	ICA73,	but	shares	the	essential	alanine	and	phenylalanine	residues	identified	in	our	study	(Brueggemann	et	al.,	2014).	This	is	further	confirmed	since	substituting	these	two	residues	into	KCNQ3	only	partially	endowed	ICA73-sensitivity.		Taken	 together,	 our	 findings	 demonstrate	 that	 despite	 sharing	 structural	similarities	with	retigabine,	ICA73	acts	via	an	entirely	different	mechanism	of	action.	Specific	 residues	 distant	 from	 the	 putative	 retigabine	 binding	 site	 are	 able	 to	influence	ICA73	effects	on	KCNQ2,	and	underlie	subtype	specificity	of	 the	drug	 for	KCNQ2	over	KCNQ3.	 	Moreover,	mutations	of	 these	 residues	 in	 the	voltage	sensor	domain	have	multi-pronged	effects	on	ICA73	actions,	as	some	are	able	to	specifically	abolish	 the	 effects	 of	 ICA73	 on	 voltage-dependence,	 while	 preserving	 current			 48	potentiation.	Our	findings	demonstrate	unambiguously	that	KCNQ	openers	should	be	classified	into	at	least	two	subgroups	based	on	their	primary	site	of	action	(which	can	be	 determined	 based	 on	 differential	 effects	 of	 the	 KCNQ2[W236L/F]	 or	 [F168L]	mutations).	 Ongoing	 investigation	 of	 the	 multi-pronged	 effects	 of	 KCNQ	 openers	acting	 on	 the	 voltage	 sensor	will	 hopefully	 lead	 to	 a	 deeper	 understanding	 of	 the	general	principles	underlying	the	actions	of	these	drugs.			 49	Chapter	 4:	 In-depth	 Analysis	 of	 Voltage	 Sensor	 Residues	Involved	in	ICA73	Effects	on	KCNQ2	4.1	Introduction		 With	 the	 emergence	 of	 potassium	 channel	 activators	 as	 a	 novel	 class	 of	potential	drugs	 for	 treating	neuronal	and	smooth	muscle	disorders,	 it	 is	becoming	apparent	that	there	are	at	least	two	classes	of	compounds	that	act	through	distinct	mechanisms	on	the	KCNQ	M-channel	family.	While	the	only	approved	drugs	on	the	market,	 retigabine	 and	 flupirtine,	 have	 been	 shown	 recently	 to	 act	 through	 a	hydrogen-bond	with	the	conserved	S5	Trp	residue	in	KCNQ2-5	channels	(Kim	et	al.,	2015),	 various	other	 compounds	have	been	 found	 to	 act	 in	 a	different	manner.	 In	particular,	 a	 few	compounds	 from	 the	diclofenac	 family	and	pyridinyl	benzamides	manufactured	by	Icagen	have	been	shown	to	act	independently	of	the	S5	Trp	residue,	likely	 acting	 through	 the	 VSD	 instead	 (Padilla	 et	 al.,	 2009;Peretz	 et	 al.,	 2010).	However,	there	has	been	very	little	understanding	of	potential	binding	residues	in	the	VSD,	particularly	in	terms	of	residues	that	may	allow	some	drugs	to	exhibit	subtype	specificity.		In	Chapter	3,	we	demonstrated	that	ICA73	exhibits	strong	subtype	specificity,	activating	 KCNQ2	 but	 not	 KCNQ3	 channels,	 and	 we	 investigated	 the	 structural	determinants	of	this	specificity.	Using	a	chimeric	approach,	we	pinpointed	the	site	of	action	of	ICA73	to	be	in	the	VSD	and	focused	on	the	S3-S4	region	since	swapping	the	KCNQ3	 S3-S4	 region	 into	 KCNQ2	 completely	 abolished	 ICA73	 effect.	 Screening	mutagenesis	allowed	us	to	identify	two	residues	in	the	S3	segment	of	KCNQ2,	F168			 50	and	 A181,	 that	 strongly	 influence	 the	 subtype	 specificity	 of	 ICA73	 effects.	Importantly,	 substituting	 these	 residues	 back	 into	 KCNQ3	 (L198F	 and	 P211A),	respectively,	endowed	ICA73	sensitivity	to	KCNQ3	channels.	While	our	electrophysiology	techniques	allowed	us	to	determine	the	specific	residues	 involved	 in	 ICA73	 effect,	 it	 is	 difficult	 to	 distinguish	 residues	 directly	involved	 in	 binding	 from	 those	 that	 are	 involved	 in	 transducing	 binding	 to	 a	functional	effect.	Unexpectedly,	we	noticed	that	there	are	two	separable	effects	that	ICA73	exerts	on	KCNQ2	channels.	First,	the	drug	induces	a	dramatic	hyperpolarizing	gating	shift.	Second,	ICA73	causes	increased	current	magnitude	by	approximately	at	least	2-fold,	at	the	plateau	of	the	conductance-voltage	relationship.	The	separation	of	these	 two	outcomes	 is	 evident	 in	 the	KCNQ2[A181P]	 channel,	where	 the	 leftward	shift	 by	 ICA73	 in	 the	 conductance-voltage	 relationship	 is	 minimal	 but	 current	potentiation	is	preserved.		In	 this	 chapter,	 I	 sought	 to	 investigate	 ICA73	 interactions	with	 the	 voltage	sensor	 in	 more	 detail.	 We	 revisited	 the	 S1-S2	 region	 of	 the	 VSD	 since	 the	 Q2	 +	Q3[S1,S2]	 chimera	 had	 a	 similar	 profile	 as	 KCNQ2[A181P],	 using	 a	 scanning	mutagenesis	approach.	Although	mutagenesis	in	this	region	did	not	identify	further	residues	that	abolish	ICA73	effects,	we	noticed	several	residues	that	either	altered	native	channel	function	or	specific	aspects	of	ICA73	effects.	In	addition,	we	observed	further	 evidence	 for	 separable	 effects	 of	 ICA73.	 Finally,	 we	 investigated	 KCNQ2	residue	K219.	This	position	was	highlighted	briefly	in	Chapter	3,	because	molecular	models	 of	 KCNQ2	 open	 and	 closed	 states	 predict	 that	 K219	 is	 conformationally	mobile	and	comes	into	close	proximity	with	KCNQ2	F168	in	the	channel	open	state.			 51	Our	findings	suggest	an	important	role	for	this	residue	in	channel	opening,	and	may	lead	to	further	investigations	of	how	this	fits	into	the	ICA73	actions.	While	we	were	unable	to	single	out	residues	that	are	responsible	for	ICA73	specificity	in	this	chapter,	the	 experiments	 have	 given	 us	 insight	 on	 overall	 channel	 function	 and	 laid	 the	foundation	for	future	studies.	4.2	Results	4.2.1	Characterizing	the	Influence	of	the	S1-S2	Segments	on	ICA73	Sensitivity	In	 Chapter	 3,	 we	 highlighted	 that	 the	 KCNQ2[A181P]	 mutant	 channel	exhibited	 an	 unexpected	 selective	 reduction	 of	 the	 ICA73-mediated	 gating,	 while	preserving	drug	effects	on	current	potentiation.	This	phenotype	was	also	observed	in	our	 initial	 electrophysiological	 characterization	 of	 the	 Q2+Q3[S1,S2]	 chimeric	channel.	Therefore,	we	have	undertaken	a	more	detailed	characterization	of	the	S1	and	S2	segments	in	terms	of	their	influence	on	ICA73	effects.	We	noted	that	the	S1	and	S2	segments	of	KCNQ2	and	KCNQ3	diverged	significantly,	whereas	the	S3	and	S4	segments	 had	 very	 few	 sequence	 differences.	 Aiming	 to	 possibly	 narrow	 down	essential	 sequences	 for	 normal	 ICA73	 sensitivity,	 we	 constructed	 more	 specific	chimeras	by	swapping	only	the	S1	or	S2	segment	of	KCNQ2	and	KCNQ3.	We	observed	that	both	of	these	chimeric	channels	retain	ICA73	sensitivity	(Fig.	4-1B,C).	However,	the	Q2	+	Q3[S2]	construct	exhibited	a	marked	reduction	in	the	hyperpolarizing	shift	by	ICA73	while	preserving	the	current	potentiation.				 52						Figure	4-1	S1-S2	domain	and	ICA73	sensitivity.	Conductance-voltage	relationships	for	(A)	Q2	+	Q3[S1-S2]	(control	V1/2	=	-9	±	1	mV,	k	=	8	±	1	mV;	ICA73	V1/2	=	-22	mV	±	5	mV,	k	=	5	±	1	mV,	n	=	5),	(B)	Q2	+	Q3[S1]	(control	V1/2	=	-9	±	2	mV,	k	=	13	±	1	mV,	n	=	4),	and	(C)	Q2	+	Q3[S2]	(control	V1/2	=	-27	±	1	mV,	k	=	18	±	4	mV,	n	=	5;	 ICA73	V1/2	=	-62	±	3	mV,	k	=	7	±	1	mV,	n	=	4)	normalized	to	peak	conductance	in	control.	(D)	Sequence	alignment	between	KCNQ2	and	KCNQ3	in	S1-S2	region.	In	all	panels,	error	bars	represent	s.e.m.		4.2.2	ICA73	Effect	on	S1	Point	Mutations		Despite	the	Q2	+	Q3[S1]	construct	appearing	to	show	full	sensitivity	to	ICA73	(Fig.	 4-1B),	 we	 investigated	 the	 effects	 of	 substituting	 of	 individual	 residues	 (or	clusters	of	neighbouring	residues)	from	KCNQ3	into	KCNQ2	(Fig.	4-2).	As	one	might	expect,	 most	 of	 the	 mutants	 were	 quite	 responsive	 to	 ICA73,	 exhibiting	 a	hyperpolarizing	 gating	 shift	 as	 well	 as	 current	 potentiation.	 However,	 certain	noteworthy	 differences	 stood	 out	 at	 some	 positions.	 Some	 residue	 substitutions	exhibited	 a	 less	 dramatic	 gating	 shift	 relative	 to	WT	 KCNQ2	 (eg.	 S110A,	 S105G),			 53	highlighted	 by	 near	 complete	 channel	 closure	 at	 hyperpolarizing	 voltages.	 Other	mutants	(eg.	Y98L)	exhibited	significantly	greater	potentiation	relative	to	WT	KCNQ2.	Interestingly,	 the	 KCNQ2[I115F]	 construct	 (Fig.	 4-2H)	 exhibited	 a	 clear	hyperpolarizing	shift	by	ICA73	but	a	much	smaller	current	potentiation	effect	relative	to	WT	KCNQ2.			Figure	4-2	Characterizing	S1	point	mutations	and	ICA73	effect.	(A-H)	Conductance-voltage	relationships	for	(A)	Q2	F93L/I94L	(control	V1/2	=	-30	±	8	mV,	k	=	12	±	1	mV,	n	=	3),	(B)	Q2	Y98L	(control	V1/2	=	-33	±	4	mV,	k	=	10	±	2	mV,	n	=	3),	(C)	Q2	L102I	(control	V1/2	=	-38	±	3	mV,	k	=	14	±	1	mV,	n	=	5),	(D)	Q2	S105G	(control	V1/2	=	-27	±	1	mV,	k	=	9	±	1	mV,	n	=	3),	(E)	Q2	V108I	(control	V1/2	=	-31	±	6	mV,	k	=	12	±	1	mV,	n	=	5),	(F)	Q2	S110A	(control	V1/2	=	-21	±	8	mV,	k	=	12	±	1	mV,	n	=	4),	(G)	Q2	F112L/S113T	(control	V1/2	=	-26	-±	4	mV,	k	=	13	±	2	mV,	n	=	3),	and	(H)	Q2	 I115F	 (control	V1/2	=	 -42	±	 8	mV,	 k	 =	 12	±	 2	mV,	 n	 =	 4)	 normalized	 to	 peak	conductance	 in	 control.	 (I)	Molecular	model	 of	 voltage-sensing	 domain	 of	 KCNQ2	channels	highlighting	S1	residues	screened	(yellow)	and	 I115	(blue).	 In	all	panels,	error	bars	represent	s.e.m.				 54	4.2.3	ICA73	Effect	on	S2	Point	Mutations	We	also	investigated	the	S2	segment	because	the	Q2	+	Q3[S2]	chimera	had	a	reduced	 response	 to	 ICA73	 (Fig.	 4-1C).	 Again,	 we	 screened	 each	 position	 where	KCNQ2	 and	 KCNQ3	 differed.	 To	 our	 surprise,	 none	 of	 these	 mutations	 severely	hindered	the	ICA73	effect	(Fig.	4-3),	although	there	were	some	notable	consequences	of	 some	mutants.	 The	KCNQ2[T133A]	 (Fig.	 4-3H)	mutant	 had	 a	 very	 right-shifted	conductance-voltage	relationship	relative	to	WT	KCNQ2	and	other	mutant	constructs,	but	both	features	of	the	ICA73	effect	persisted.	Also,	the	KCNQ2[G124D]	(Fig.	4-3C)	mutant	channel	exhibited	much	greater	current	potentiation.	Overall,	based	on	the	sequence-motivated	electrophysiological	screening	of	S1	and	S2	mutants,	we	were	unable	 to	 find	 specific	 residues	 essential	 for	 ICA73	 sensitivity,	 with	 the	 possible	exception	of	KCNQ2[I115F]	(Fig.	4-2H).	Further	experiments	are	necessary	to	look	at	different	 aspects	 the	 mutations	 may	 have	 on	 channel	 function;	 for	 example,	comparing	 V1/2	 values	 to	 amount	 of	 shift	 in	 conductance-voltage	 relationships	 or	magnitude	of	current	potentiation.	At	this	point,	it	appears	that	the	two	residues	in	S3,	 F168L	 and	 A181P,	 as	 discussed	 in	 Chapter	 3,	 are	 the	 ones	 that	 specifically	influence	ICA73	effect.			 55		Figure	4-3	Characterizing	S2	point	mutations	and	ICA73	effect.	(A-K)	Conductance-voltage	relationships	for	(A)	Q2	K120T/S121V	(control	V1/2	=	-35	±	5	mV,	k	=	12	±	1	mV,	n	=	5),	(B)	Q2	E123G	(control	V1/2	=	-26	±	6	mV,	k	=	12	±	1mV,	n	=	3),	(C)	Q2	G124D	(control	V1/2	=	-22	±	5	mV,	k	=	14	±	1	mV,	n	=	3),	(D)	Q2	A125W	(control	V1/2	=	-22	±	7	mV,	k	=	11	±	1	mV,	n	=	3),	(E)	Q2	Y127L/I128L	(control	V1/2	=	-20	±	3	mV,	k	=	11	±	1	mV,	n	=	3),	(F)	Q2	I131T	(control	V1/2	=	-32	±	2	mV,	k	=	10	±	1	mV,	n	=	3),	(G)	Q2	V132F	(control	V1/2	=	-29	±	9	mV,	k	=	10	±	1	mV,	n	=	3),	(H)	Q2	T133A	(control	V1/2	=	2	±	2	mV,	k	=	21	±	1	mV,	n	=		3),	(I)	Q2	V135F/V136I	(control	V1/2	=	-28	±	4	mV,	k	=	14	±	2	mV,	n	=	5),	(J)	Q2	V139A	(control	V1/2	=	-25	±	2	mV,	k	=	11	±	2	mV,	n	=	5),	and	(K)	Q2	Y141F/F142A/V143L	(control	V1/2	=	-36	±	1	mV,	k	=	11	±	1	mV,	n	=	3)	normalized	to	peak	conductance	in	control.	(L)	Molecular	model	 of	 voltage-sensing	 domain	 of	 KCNQ2	 channels	 highlighting	 S2	 residues	screened	(yellow),	G124	(blue),	and	T133	(blue).	In	all	panels,	error	bars	represent	s.e.m.			 			 56	4.2.4	Potential	Binding	Pocket	Residues	and	ICA73	Effects	Since	our	mutagenesis	and	electrophysiology	experiments	cannot	distinguish	between	the	binding	vs.	effect	of	ICA73	on	KCNQ2,	we	were	interested	in	deciphering	the	 possible	 binding	 site	 of	 ICA73.	 Molecular	 simulations	 of	 solvent	 accessible	pockets	 in	 the	KCNQ2	 structure	highlighted	 several	 potential	 binding	 residues	 for	ICA73	 (Fig.	 4-4).	We	mutated	 each	 of	 these	 residues	 to	 alanine	 and	 tested	 ICA73	effects	(Fig.	4-4).	The	Q2	R201A	mutation	(Fig.	4-4H)	resulted	in	a	channel	that	was	almost	 constitutively	 open	 in	 the	 absence	 of	 ICA73;	 addition	 of	 ICA73	 further	enhanced	the	opening	of	the	channel	at	very	hyperpolarizing	potentials,	but	did	not	dramatically	potentiate	 currents.	This	effect	may	arise	because	R201	 is	one	of	 the	crucial	 positive	 charges	 in	 the	 voltage	 sensor,	 thus	 neutralizing	 the	 charge	 at	 this	position	may	destabilize	the	resting	state	of	the	voltage	sensor,	allowing	the	channel	to	open	with	little	voltage-dependence.	The	dramatic	relative	stabilization	of	the	open	state,	 even	 in	 the	 absence	 of	 ICA73,	 likely	 underlies	 the	 weaker	 ICA73-mediated	potentiation	observed	in	the	R201A	mutant	channel.	More	interestingly,	we	observed	a	 second	point	mutation	 that	 duplicates	 the	 effects	 of	 the	KCNQ2[A181P]	mutant.	Specifically,	the	KCNQ2[E130A]	mutation	(Fig.	4-4C)	almost	completely	abolished	the	ICA73-mediated	hyperpolarizing	gating	shift,	but	retained	current	potentiation.				 57			Figure	4-4	ICA73	effect	on	potential	binding	pocket	residues.	(A-I)	Conductance-voltage	relationships	for	(A)	Q2	C106A	(control	V1/2	=	-43	±	7	mV,	k	=	16	±	1	mV,	n	=	3),	(B)	Q2	L107A	(control	V1/2	=	5	±	2mV,	k	=	14	±	1	mV,	n	=	3),	(C)	Q2	E130A	(control	V1/2	=	-8	±	3mV,	k	=	15	±	2	mV,	n	=	4;	ICA73	V1/2	=	-39	±	3	mV,	k	=	11	±	1	mV,	n	=	4),	(D)	Q2	I134A	(control	V1/2	=	-52	±	1	mV,	k	=	14	±	1	mV,	n	=	3),	(E)	Q2	S179A	(control	V1/2	=	-16	±	1	mV,	k	=	18	±	2	mV,	n	=	3),	(F)	Q2	V182A	(control	V1/2	=	1	±	7	mV,	k	=	15	±	2	mV,	n	=	3),	(G)	Q2	R198A	(control	V1/2	=	-9	±	1	mV,	k	=	10	±	1	mV,	n	=	3),	(H)	Q2	R201A	(n	=	5),	and	(I)	Q2	Q204A	(control	V1/2	=	-32	±	3	mV,	k	=	10	±	2	mV,		n	=	3)	normalized	to	peak	conductance	in	control.	In	all	panels,	error	bars	represent	s.e.m.	(J)	Molecular	model	of	KCNQ2	channel	showing	potential	binding	pocket	residues	 (yellow)	and	highlighting	E130	(blue)	and	R201	(blue).			 			 58	4.2.5	KCNQ2	I115F	may	have	Reduced	Current	Potentiation	by	ICA73	As	 mentioned	 above,	 the	 KCNQ2[I115F]	 construct	 appeared	 to	 exhibit	 a	significantly	 reduced	 ICA73-mediated	 currently	 potentiation,	 and	 we	 have	highlighted	this	experimental	finding	in	Fig.	4-5.	We	consistently	observed	very	small	increases	in	current	magnitude	with	addition	of	ICA73	relative	to	WT	KCNQ2	(Fig.	5-5A,B,F,G).	We	have	included	cell-by-cell	plots	of	current	magnitude	at	20	mV	before	and	 after	 drug	 for	 both	WT	 KCNQ2	 (Fig.	 5-5C)	 and	 KCNQ2[I115F]	 (Fig.	 5-5D)	 to	illustrate	this	trend.	Located	at	the	top	of	the	S1-S2	linker	(Fig.	5-5E),	position	115	may	play	a	role	in	facilitating	the	current	potentiation	we	observe	with	ICA73	on	most	KCNQ2	constructs.	As	illustrated	in	Shaker,	there	is	a	link	between	the	pore	region	and	 S1	 that	may	 be	 an	 important	 determinant	 of	 coupling	 between	 the	 pore	 and	voltage	sensor	(Lee	et	al.,	2009b).	Further	studies	need	to	be	conducted	to	clarify	and	validate	this	observation.				 59		Figure	4-5	Current	potentiation	by	ICA73	may	be	reduced	in	KCNQ2[I115F].	(A,B)	Conductance-voltage	relationships	of	(A)	KCNQ2	wild	type	(control	V1/2	=	-40	±	5	mV,	k	=	5	±	1	mV,	n	=	5)	and	(B)	KCNQ2[I115F]	(control	V1/2	=	-42	±	8	mV,	k	=	12	±	 2	mV,	 n	 =	 4)	 normalized	 to	 peak	 conductance	 in	 control.	 (C,D)	 Plots	 of	 current	magnitude	at	20	mV	with	and	without	30	µM	ICA73	of	sample	individual	cells	for	(C)	KCNQ2	wild	type	and	(D)	KCNQ2[I115F].	(E)	Molecular	model	of	top	view	of	voltage-sensing	 domain	 highlighting	 I115	 (blue).	 (F,G)	 Exemplar	 patch	 clamp	 current	recordings	of	KCNQ2[I115F]	in	(F)	control	and	(G)	with	30	µM	ICA73.			 			 60	4.2.6	 KCNQ2[E130A]	 Abolishes	 the	 ICA73-mediated	 Shift	 but	 not	 Current	Potentiation	One	 of	 the	 suggested	 potential	 binding	 pocket	 residues,	 E130	 is	 a	 highly	conserved	residue	among	voltage-gated	potassium	channels	 that	acts	as	a	voltage-sensor	counter	charge	(Fig	4-6E)	(Papazian	et	al.,	1995;Tao	et	al.,	2010;Pless	et	al.,	2011;Pless	 et	 al.,	 2014).	 Previous	 studies	 have	 suggested	 that	mutations	 at	 E130	abolished	 the	 effects	 of	 ICA73	 and	 closely-related	 compounds	 (Li	 et	 al.,	2013b;Brueggemann	 et	 al.,	 2014).	 However,	 our	 study	 carefully	 distinguished	 the	gating	shift	and	potentiation	effects	of	ICA73,	revealing	that	while	the	hyperpolarizing	shift	 by	 ICA73	 is	 mostly	 eliminated,	 the	 current	 potentiation	 is	 still	 present	 in	KCNQ2[E130A]	channels	(Fig.	4-6A,B).		As	mentioned	in	Chapter	3,	this	is	a	feature	of	the	 data	 that	 appears	 to	 have	 often	 been	 overlooked	 due	 to	 common	practices	 of	normalizing	 conductance-voltage	 relationships	 to	 peak	 current	 in	 the	 given	condition.	Looking	at	the	kinetics	of	this	construct,	we	also	notice	that	in	the	presence	of	ICA73,	channel	closure	is	decelerated	(Fig.	4-6C),	suggesting	that	ICA73	most	likely	still	 binds	 to	 the	 channel,	 but	 the	 mutation	 alters	 the	 structure	 in	 a	 way	 that	eliminates	the	hyperpolarizing	shift	of	voltage-dependence.									 61		Figure	4-6	ICA73-mediated	shift	is	abolished	in	KCNQ2[E130A]	which	preserves	current	potentiation.6	(A)	Exemplar	patch	clamp	current	recordings	of	KCNQ2[E130A].	(B)	Conductance-voltage	relationship	of	KCNQ2[E130A]	(control	V1/2	=	-8	±	3mV,	k	=	15	±	2	mV,	n	=	4;	ICA73	V1/2	=	-39	±	3	mV,	k	=	11	±	1	mV,	n	=	4)	normalized	to	peak	conductance	in	control.	 (C)	Time	 constants	 (τ)	 of	 channel	 closure	were	measured	by	pulsing	 to	 a	range	 of	 negative	 voltages	 from	 a	 holding	 potential	 of	 +20	mV;	 inset,	 sample	 tail	currents	 of	 KCNQ2[E130A]	 at	 -130	 mV.	 (D)	 Sequence	 alignment	 highlighting	conserved	E130	in	numerous	Kv	channels.								 62	4.2.7	KCNQ2	K219	may	Serve	Important	Role	in	Channel	Opening	Based	on	our	previous	finding	that	the	KCNQ2[F168L]	mutant	is	completely	unresponsive	to	ICA73,	we	investigated	the	role	of	residue	K219	that	is	positioned	in	close	 proximity	 to	 the	 F168	 in	 the	 open	 state	 of	 KCNQ2.	 Moreover,	 in	 molecular	models	of	KCNQ2	activation,	there	is	considerable	motion	of	K219	relative	to	F168,	with	these	two	sidechains	only	suggested	to	approach	one	another	very	late	in	the	activation	sequence.	We	hypothesized	that	the	positive	charge	of	K219	may	form	a	stabilizing	interaction	with	the	π-electrons	in	the	aromatic	ring	of	Phe.	Mutating	K219	to	 either	 an	 alanine	 or	 arginine	 significantly	 perturbed	 channel	 function;	 current	magnitude	 in	 control	 conditions	 was	 very	 small	 for	 both	 mutants	 (Fig.	 4-7A,B).	Interestingly,	 ICA73	 addition	 caused	 dramatic	 potentiation	 of	 current	 in	 both	KCNQ2[K219A]	and	[K219R]	(Fig.	4-7A,B).	While	arginine	is	also	positively	charged,	we	speculate	that	the	charge	density	on	the	lysine	may	be	optimal	for	the	stabilizing	interaction	 and	 thus	 the	 K219R	 mutant	 behaves	 similarly	 to	 the	 K219A	 mutant.	Additional	experiments	should	be	conducted	to	elucidate	the	specific	role	of	K219	in	terms	of	interactions	with	F168	and	ICA73	effects.					 63			Figure	4-7	Examining	the	potential	role	of	KCNQ2	K219	in	channel	opening.	(A,B)	Exemplar	patch	clamp	current	recordings	of	KCNQ2[K219A]	in	(A)	control	and	(B)	 with	 30	 µM	 ICA73.	 (D,E)	 Exemplar	 patch	 clamp	 current	 recordings	 of	KCNQ2[K219R]	in	(D)	control	and	(E)	with	30	µM	ICA73.	(C,F)	Conductance-voltage	relationships	of	(C)	KCNQ2[K219A]	(control	V1/2	=	-3	±	1	mV,	k	=	12	±	2,	n	=	3)	and	(F)	KCNQ2[K219R]	(control	V1/2	=	-21	±	1	mV,	k	=	11	±	2	mV,	n	=	3)	normalized	to	peak	conductance	in	control.	(G)	open	and	closed	states	of	KCNQ2	highlighting	K219	and	F168	in	bottom	and	side	views.				 64	4.3	Discussion	There	is	growing	recognition	of	the	therapeutic	potential	of	potassium	channel	activators	on	neuronal	diseases	such	as	pain	and	epilepsy	as	well	as	smooth	muscle	diseases.	 As	 the	 mechanisms	 of	 action	 of	 retigabine	 and	 flupirtine	 have	 been	unravelled,	there	is	more	interest	in	improving	the	design	of	this	drug	class	for	better	specificity.	However,	retigabine	and	flupirtine	remain	the	only	KCNQ	activators	(or	Kv	 channel	 activators)	 approved	 for	 widespread	 human	 use.	 Certain	 compounds	including	ICA73	have	shown	strong	subtype	specificity.	In	the	previous	chapter,	we	demonstrated	 through	 chimeric	 and	mutagenesis	 approaches	 that	 ICA73	 interacts	with	the	VSD	rather	than	the	retigabine	binding	site	in	the	pore	region.	Particularly,	two	residues	in	the	S3	of	KCNQ2,	F168	and	A181,	are	important	for	ICA73	specificity.	Mutations	 at	 these	positions	 significantly	 reduce	drug	effect	 on	 the	 channel.	More	importantly,	 we	 showed	 that	 the	 ICA73	 effect	 has	 two	 components	 that	 may	 be	separable:	 first,	 a	 hyperpolarizing	 gating	 shift,	 and	 second,	 an	 increase	 in	 current	magnitude.	In	 this	 chapter,	we	 further	 investigated	different	 regions	of	 the	KCNQ2	and	KCNQ3	voltage	sensor,	in	order	to	better	understand	how	ICA73	interacts	with	and	influences	KCNQ2.	We	performed	a	detailed	analysis	of	the	S1-S2	region	of	the	VSD,	motivated	by	chimeric	studies	showing	that	replacement	of	the	KCNQ2[S1-S2]	with	corresponding	sequence	of	KCNQ3	abolished	the	ICA73-mediated	gating	shift.	After	screening	each	residue	that	differs	between	KCNQ2	and	KCNQ3,	we	found	none	that	altered	the	ICA73	effect	substantially.	Since	the	S1-S2	region	in	KCNQ2	and	KCNQ3			 65	differ	 significantly,	 the	 chimeric	 construct	 Q2	 +	 Q3[S1,S2]	 may	 have	 an	 altered	structure	which	results	in	the	differential	response	to	ICA73.		Although	 the	S1-S2	screen	did	not	 suggest	any	specific	 residues	 that	 ICA73	may	interact	with,	we	noticed	that	KCNQ2[I115F]	may	have	a	weakened	response	to	ICA73.	While	 ICA73	 induces	a	pronounced	hyperpolarizing	shift	 to	KCNQ2[I115F],	there	appears	to	be	minimal	potentiation	of	currents.	This	is	distinct	from	what	we	reported	 for	 the	KCNQ2[A181P]	mutant,	 again	 demonstrating	 separable	 effects	 of	ICA73.	Data	on	 this	particular	observation	 is	preliminary	but	 it	 is	 likely	 that	more	experiments	can	tease	out	the	importance	of	the	I115	residue	and	its	possible	role	in	facilitating	current	potentiation	by	ICA73.	We	believe	that	its	location	at	the	top	of	the	S1-S2	linker	in	the	extracellular	domain	may	contribute	to	this	role	(Lee	et	al.,	2009b).		To	 further	 deduce	 the	 effects	 of	 ICA73	 and	 its	 possible	 interactions	 with	KCNQ2	 channels,	 we	 examined	 the	 KCNQ2	 structural	model,	 identifying	 potential	residues	that	make	up	solvent	accessible	cavities	where	ICA73	may	bind.	We	noted	that	all	of	these	residues	are	found	in	both	KCNQ2	and	KCNQ3,	and	many	are	highly	conserved	among	voltage-gated	potassium	channels.	Thus,	mutating	some	of	these	residues	to	alanine	altered	the	channel	function.	For	example,	KCNQ2[R201A]	lost	its	sigmoidal	conductance-voltage	relationship	as	 the	channel	was	essentially	open	at	very	 hyperpolarized	 potentials	 even	 in	 control	 conditions.	 However,	 addition	 of	ICA73	still	increased	the	magnitude	of	current	of	this	construct,	suggesting	that	the	mutation	 likely	 did	 not	 damage	 the	 ICA73	 interaction	 with	 the	 channel.	 We	 also	identified	KCNQ2[E130A]	which	was	a	mutation	shown	in	other	studies	to	abolish	ICA73	effect	(Li	et	al.,	2013b;Brueggemann	et	al.,	2014).	However,	we	observed	that			 66	current	 potentiation	 was	 preserved,	 a	 feature	 which	 had	 not	 been	 discussed	 in	previous	studies.	Given	these	persistent	ICA73-mediated	effects,	we	do	not	attribute	this	 residue	as	an	essential	 ICA73	binding	 residue.	Rather,	we	suspect	 that	effects	observed	in	the	A181P	and	E130A	mutations	may	be	largely	explained	by	a	shift	in	the	state-dependence	of	ICA73	binding	(see	Chapter	5:	General	Discussion).	Finally,	we	investigated	K219,	a	residue	that	we	hypothesized	may	form	a	stabilizing	interaction	with	F168	in	the	KCNQ2	open	state.	We	proposed	that	the	positive	charge	of	 lysine	at	 this	position	played	an	 important	role,	possibly	 forming	an	 interaction	with	the	aromatic	π-electrons	of	F168.	Surprisingly,	we	recorded	little	current	from	both	 KCNQ2[K219A]	 and	 KCNQ2[K219R]	 	 in	 control	 conditions,	 far	 smaller	 than	other	 constructs	 tested;	 however,	 application	 of	 30	 µM	 ICA73	 induced	 a	 large	hyperpolarizing	 gating	 shift	 and	 a	 dramatic	 current	 increase.	While	 the	 alanine	 is	uncharged,	we	expected	the	positively	charged	arginine	to	provide	at	least	a	partial	rescue	of	the	WT	phenotype,	but	this	did	not	turn	out	to	be	the	case.	It	is	possible	that	the	delocalized	positive	 charge	 in	 the	arginine	 residue	 is	 insufficient	 to	mimic	 the	presence	of	a	lysine,	or	perhaps	the	positive	charge	on	the	lysine	is	oriented	in	a	way	that	 provides	 a	 more	 optimal	 interaction	 with	 F168.	 Future	 experiments	 should	address	this	observation	and	investigate	the	R213	residue	that	is	also	located	in	the	same	region,	possibly	interacting	with	F168.	As	more	details	on	ICA73	and	KCNQ2	unfold,	better	understanding	of	the	channel	structure	and	the	mechanism	of	action	of	ICA73	can	lead	to	improved	drug	design	with	minimal	side	effects.			 			 67	Chapter	5:	General	Discussion	Development	of	KCNQ	channel	activators:		 While	 there	 are	 over	 20	 anti-epileptic	 drugs	 on	 the	market,	 approximately	30%	 of	 patients	 are	 resistant	 to	 current	 pharmacotherapy	 (Brodie	 and	 French,	2000;Kwan	and	Brodie,	2010).	Thus,	the	development	and	approval	of	retigabine,	the	first	anti-epileptic	drug	that	acts	as	a	voltage-gated	potassium	channel	opener,	has	stirred	up	interest	in	the	therapeutic	potential	of	this	new	drug	class.	By	increasing	the	activity	of	KCNQ2/3	channels	around	the	resting	membrane	potential,	retigabine	can	have	a	hyperpolarizing	influence	on	membrane	voltage,	and	negatively	modulate	neuronal	firing	rate	(Orhan	et	al.,	2012).	However,	retigabine	can	also	activate	other	members	 of	 the	 KCNQ	 channel	 family,	 including	 KCNQ4	 and	 KCNQ5	 which	 are	prominently	expressed	in	smooth	muscle	and	the	ear	rather	than	the	brain.	This	lack	of	 specificity	 arises	 because	 retigabine’s	 site	 of	 action	 is	 a	 conserved	 Trp	 residue	present	 in	 the	 S5	 segment	 of	 KCNQ2-5	 channels.	 In	 addition	 to	 lacking	 subtype	specificity,	 retigabine	 affects	 other	 receptors	 such	 as	 GABA-receptors	 (Gribkoff,	2003;van	Rijn	and	Willems-van,	2003),	and	this	may	also	contribute	to	various	side	effects	 that	 have	 arisen.	 Patients	 have	 experienced	 recurring	 problems	 involving	urinary	retention,	dizziness,	and	blue-grey	mucocutaneous	discoloration,	to	name	a	few	(Streng	et	al.,	2004;Orhan	et	al.,	2012;	Garin	et	al.,	2014).	Therefore,	there	is	a	need	for	ongoing	improvement	of	this	first-in-class	drug.			 Numerous	drugs	with	KCNQ	channel	opener	activity	have	been	synthesized.		Many	of	the	designs	(including	ICA73	characterized	in	this	study)	are	based	on	the			 68	structure	of	retigabine.	As	more	compounds	are	tested,	it	is	becoming	apparent	that	several	may	act	differently	from	retigabine.	Rather	than	targeting	the	pore	region	of	KCNQ	channels,	it	has	been	shown	that	some	Icagen	compounds	and	NH29	from	the	diclofenac	 family	 may	 act	 on	 the	 VSD	 (Padilla	 et	 al.,	 2009;Peretz	 et	 al.,	 2010).	However,	the	mechanism	of	action	of	these	compounds	are	mostly	still	unknown.		In	 this	 concluding	 chapter,	 I	 hope	 to	 highlight	 three	 fundamental	 points	related	to	the	mechanism	of	action	of	ICA73.	First,	ICA73	has	a	‘two-pronged’	effect	on	drug	sensitive	channels:	it	causes	a	hyperpolarizing	gating	shift,	in	addition	to	a	pronounced	potentiation	of	peak	current.	These	effects	are	markedly	greater	than	the	effects	 of	 retigabine,	 but	 only	 in	 certain	KCNQ	 channel	 types	 (eg.	 KCNQ2,	 but	 not	KCNQ3).	 Second,	 ICA73	acts	on	a	unique	binding	 site,	 distinct	 from	 the	 retigabine	binding	 site.	 Lastly,	 the	 gating	 and	 potentiation	 effects	 of	 ICA73	 appear	 to	 be	‘separable’	in	the	sense	that	I	have	generated	point	mutants	that	can	exhibit	one	effect	in	the	absence	of	the	other	(particularly,	potentiation	in	the	absence	of	a	gating	shift).	I	will	review	the	evidence	and	implications	of	these	features	of	ICA73	modulation	of	KCNQ2.		Large	subtype-selective	actions	of	ICA73		 In	 Chapter	 3,	 we	 demonstrated	 that	 ICA-069673	 (ICA73),	 a	 pyridinyl	benzamide,	selectively	activates	KCNQ2	over	KCNQ3	channels.	It	shifts	the	voltage-dependence	of	activation	of	KCNQ2	channels	to	very	hyperpolarized	potentials.	The	ICA73-induced	 hyperpolarizing	 shift	 in	 the	 conductance-voltage	 relationship	 is	 so	large	that	we	cannot	fully	characterize	the	magnitude	of	this	effect,	simply	because	we	cannot	voltage-clamp	cells	to	a	sufficiently	negative	voltage	to	fully	close	channels			 69	in	the	presence	of	ICA73.	I	also	showed	that	by	normalizing	the	conductance-voltage	relationship	to	the	peak	conductance	in	control	conditions,	it	is	apparent	that	ICA73	induces	a	~2-fold	increase	in	current	magnitude	that	is	not	observed	with	retigabine.	These	marked	differences	from	retigabine	provided	an	initial	clue	that	ICA73	acts	via	a	distinct	mechanism	from	retigabine.	I	 exploited	 the	differences	between	 ICA73	effects	on	KCNQ2	and	KCNQ3	 to	investigate	the	site	of	action	of	ICA73.	A	chimeric	approach	was	used	to	first	narrow	down	the	importance	of	the	VSD,	and	particularly	the	S3-S4	region.	Next,	I	employed	mutagenesis	and	adapted	a	non-radioactive	Rb+	efflux	assay	to	screen	for	residues	essential	for	ICA73	effects	on	KCNQ2.	I	identified	two	residues,	F168	at	the	bottom	of	S3,	 and	 A181	 at	 the	 top	 of	 S3,	 which	 significantly	 reduced	 ICA73	 effects.	 These	findings	are	a	significant	step	forward,	as	they	are	the	first	specific	residues	reported	to	influence	subtype	selectivity	of	a	KCNQ	channel	activity.		Detailed	characterization	of	positions	underlying	subtype	specificity		 I	 used	 whole-cell	 patch	 clamp	 recordings	 to	 characterize	 the	 effects	 of	mutations	 at	KCNQ2	positions	 F168	 and	A181.	 The	 F168L	mutation	 rendered	 the	channel	completely	insensitive	to	ICA73.	I	also	determined	that	an	aromatic	residue	at	position	168	was	sufficient	for	ICA73	effect	since	the	KCNQ2[F168Y]	and	[F168W]	mutants	were	 both	 responsive	 to	 ICA73.	Mutation	 of	 KCNQ2	A181	 yielded	 a	 very	unexpected	phenotype,	where	the	current	magnitude	of	KCNQ2[A181P]	channel	was	potentiated	 by	 ICA73	 (similar	 to	 wild	 type	 KCNQ2),	 but	 the	 ICA73-mediated	hyperpolarizing	shift	was	abolished.	We	hypothesized	that	the	proline	residue	at	the	corresponding	position	in	KCNQ3	may	alter	the	helical	structure	of	S3,	and	thereby			 70	alter	 the	 interaction	of	 ICA73	with	 the	channel.	KCNQ2[A181G]	and	 [A181L]	were	both	responsive	to	ICA73,	suggesting	that	the	alanine	at	position	181	likely	plays	a	‘permissive’	role	for	ICA73	binding/effect,	rather	than	making	essential	contact	for	drug	 binding.	 Since	 channel	 closure	 of	 KCNQ2[A181P]	 is	 significantly	 slowed	 by	ICA73	(in	the	absence	of	a	shift	in	voltage-dependent	gating),	it	appears	that	binding	does	 occur	 but	 fails	 to	 translate	 into	 an	 effect	 on	 the	 voltage-dependent	 gating	mechanism.	Importantly,	substitution	of	these	residues	back	into	KCNQ3	(L198F	and	P211A,	 respectively),	 could	 rescue	some	 ICA73	effect	on	 the	otherwise	 insensitive	KCNQ3	channel.	However,	it	should	be	noted	that	the	hyperpolarizing	shift	was	much	smaller	than	in	KCNQ2,	and	the	current	magnitude	is	not	potentiated	in	the	presence	of	ICA73.				Scanning	the	VSD	reveals	that	ICA73-sensitivity	is	very	tolerant	of	mutation			 Although	the	two	residues	we	identified	in	the	S3	show	promising	evidence	of	being	involved	in	ICA73	sensitivity	and	subtype	specificity,	I	further	investigated	the	influence	of	VSD	mutations	to	fill	the	gap	of	unknowns	about	the	mechanism	of	action	of	ICA73.	In	Chapter	4,	I	revisited	the	S1-S2	region	of	the	VSD	to	test	whether	other	residues	may	influence	ICA73	sensitivity;	this	was	motivated	by	my	finding	that	the	Q2	+	Q3[S1,S2]	chimera	had	a	similar	behavior	towards	ICA73	as	the	KCNQ2[A181P]	mutant	(strong	current	potentiation	despite	no	gating	shift).	However,	after	thorough	screening	of	all	residues	that	differ	between	KCNQ2	and	KCNQ3	in	the	S1-S2	region,	I	could	 not	 identify	 particular	 residues	 that	 were	 important	 for	 ICA73	 specificity.	Nonetheless,	several	mutations	resulted	in	channels	that	behaved	differently	in	the	absence	 of	 ICA73,	 and	 KCNQ2[I115F]	 surprisingly	 exhibited	 a	 dramatic			 71	hyperpolarizing	 shift	 by	 ICA73,	 but	 the	 current	 potentiation	 was	 much	 smaller	compared	 to	wild	 type	KCNQ2.	This	observation	 further	 supports	our	observation	that	ICA73	exhibits	two	separable	effects	on	KCNQ2	channels.			 We	 also	 examined	 a	 potential	 binding	 site	 for	 ICA73	 based	 on	 published	molecular	 models	 and	 the	 presence	 of	 a	 solvent	 accessible	 pocket	 in	 a	 KCNQ2	structural	model	(Guiscard	Seebohm,	personal	communication).	Screening	numerous	residues	 did	 not	 yield	 any	 with	 notable	 effects	 on	 ICA73	 sensitivity.	 However,	 I	noticed	a	few	residues	that	altered	the	native	function	of	the	channel	(in	the	absence	of	drug).	For	example,	KCNQ2[R201A]	was	constitutively	open	even	in	the	absence	of	drug,	most	likely	due	to	the	lack	of	positive	charge	liberating	the	voltage	sensor.	In	addition,	 KCNQ2[E130A],	 a	 mutation	 previously	 reported	 to	 abolish	 the	 effect	 of	ICA73	and	similar	compounds	(Li	et	al.,	2013b;Brueggemann	et	al.,	2014),	was	found	to	abolish	 the	 ICA73	hyperpolarizing	shift,	while	 retaining	current	potentiation	by	ICA73.	This	was	an	important	result	because	it	suggests	that	the	E130A	mutation	does	not	cause	the	channel	to	become	completely	unresponsive	to	ICA73	and	that	binding	may	 very	 well	 still	 occur.	 Since	 E130	 is	 a	 highly	 conserved	 residue	 among	 most	voltage-gated	 potassium	 channels,	 acting	 as	 a	 voltage	 sensor	 counter-charge	(Papazian	et	al.,	1995;Tao	et	al.,	2010;Pless	et	al.,	2011;Pless	et	al.,	2014),	neutralizing	the	 charge	 alters	 the	 channel	 function	 and	may	 indirectly	 influence	 ICA73	 effects.	Also,	this	residue	cannot	account	for	ICA73	specificity	for	KCNQ2	(KCNQ3	channels	share	this	residue,	as	do	most	other	Kv	channels).			 My	 experimental	 characterization	 of	 the	 KCNQ2[E130A]	 highlights	 an	important	 shortcoming	 of	 many	 studies	 investigating	 KCNQ	 channel	 openers.			 72	Specifically,	there	has	been	a	common	practice	of	normalizing	conductance-voltage	relationships	(see	(Padilla	et	al.,	2009;Peretz	et	al.,	2010;Boehlen	et	al.,	2013;Li	et	al.,	2013b)),	leading	to	an	incomplete	representation	of	the	full	effect	of	the	drugs	(and	the	impact	of	mutations).	For	example,	the	KCNQ2	[E130A]	mutation	has	previously	been	reported	to	abolish	ICA73	sensitivity	(Li	et	al.,	2013b;Brueggemann	et	al.,	2014),	but	my	findings	clearly	illustrate	that	at	least	certain	aspects	of	ICA73	effects	persist	in	these	channels.	I	would	argue	that	the	drug	must	still	be	binding	to	KCNQ2[E130A]	channels,	but	that	the	effects	of	drug	binding	are	not	translated	normally	to	the	gating	machinery	of	the	channel.		 In	addition,	I	investigated	the	K219	residue	in	KCNQ2,	since	molecular	models	suggest	that	it	comes	in	close	contact	with	the	F168	residue	during	the	channel	open	state.	Whole-cell	patch	clamp	experiments	of	KCNQ2[K219A]	and	[K219R]	showed	interesting	results	in	which	channels	yielded	minimal	current	in	control	conditions,	but	 were	 dramatically	 potentiated	 upon	 addition	 of	 ICA73.	 Although	 these	observations	suggest	that	an	interaction	between	F168	and	K219	is	not	essential	for	ICA73	sensitivity,	 these	residues	may	play	an	 important	role	 in	regulation	channel	activity	Overall	significance	and	future	directions:		 Taken	 together,	 findings	 presented	 in	 Chapters	 3	 and	 4	 demonstrate	 an	extensive	 analysis	 of	 a	 potassium	 channel	 opener,	 ICA73,	 with	 a	 distinct	 activity	profile	from	retigabine.	I	utilized	the	unique	properties	of	this	compound	to	decipher	the	 differences	 between	 KCNQ2	 and	 KCNQ3	 channels,	 which	 may	 lead	 to	 better	understanding	of	subtype	differences	within	the	KCNQ	family.	My	studies	are	the	first			 73	to	define	individual	residues	that	underlie	subtype	specific	effects	of	KCNQ	channel	openers.	 I	 also	 demonstrate	 unambiguously	 that	 ICA73	 (and	 likely	 other	 closely	related	openers)	act	via	a	wholly	different	binding	site	and	mechanism	than	the	more	widely	studied	KCNQ	opener	retigabine.	Lastly,	I	have	demonstrated	that	the	effects	of	ICA73	(gating	shift,	and	potentiation)	can	be	dissociated	with	certain	mutations	in	the	 VSD,	 and	 this	 likely	 has	 important	 consequences	 for	 understanding	 the	mechanism	of	action	of	KCNQ	channel	openers.		Extending	beyond	these	results,	there	are	still	numerous	questions	remaining	that	should	be	pursued.	Firstly,	the	stoichiometry	of	ICA73	binding	to	KCNQ	channels	is	unknown,	and	could	be	 investigated	by	engineering	 tetrameric	KCNQ2	channels	with	 varying	numbers	 of	 subunits	 carrying	 either	 the	 F168L	or	A181P	mutations.	These	experiments	would	allow	us	to	study	the	stoichiometry	and	cooperativity	of	drug	effect;	for	example,	is	one	molecule	of	ICA73	acting	on	one	wild	type	monomer	of	KCNQ2	sufficient	to	generate	the	full	ICA73	effect,	or	are	four	molecules	required	to	bind	 for	a	 full	drug	effect?	 It	would	be	 interesting	 to	compare	 this	 result	 to	 the	stoichiometry	of	retigabine	on	KCNQ3	channels,	which	may	provide	further	insights	into	differences	between	ICA73	and	retigabine	actions	on	KCNQ2	channels.			 As	well,	more	investigation	of	the	KCNQ2	I115	residue	could	help	answer	the	questions	of	how	ICA73	potentiates	KCNQ2	currents,	a	 trait	not	reproducibly	seen	with	 retigabine.	One	possibility	 is	 that	 the	 location	of	 I115	at	 the	 top	of	 the	S1-S2	linker	 serves	 an	 important	 role	 in	 coupling	 the	 voltage	 sensor	 to	 the	 pore,	 as	suggested	previously	(Lee	et	al.,	2009b).	 Identifying	 the	potential	role	 this	residue	may	play	in	facilitating	current	potentiation	by	ICA73	can	again	help	us	understand			 74	the	mechanism	of	action	of	ICA73.		Similarly,	it	would	be	worth	examining	the	K219	and	R213	residues	which	may	play	crucial	roles	in	interacting	with	F168	to	facilitate	channel	opening	that	can	be	reinforced	by	ICA73.		 Finally,	the	two	separable	effects	of	ICA73	(potentiation	and	gating	shift),	has	sparked	a	hypothesis	regarding	the	potential	state-dependence	of	binding	of	ICA73.	This	appears	to	be	especially	important	for	understanding	the	unusual	conductance-voltage	relationships	of	KCNQ2[A181P],	Q2	+	Q3[S1,S2],	and	KCNQ2[E130A],	where	ICA73	 causes	 a	minimal	 gating	 shift,	 but	 dramatically	 potentiates	 currents.	 These	results	indicate	that	the	mutations	most	likely	did	not	prevent	ICA73	from	interacting	with	the	channel	(since	currents	are	still	potentiated).	However,	the	mutations	may	have	 altered	 the	 state-dependence	 of	 binding.	 We	 hypothesize	 that	 ICA73	 may	preferentially	bind	to	and	stabilize	the	activated	state	of	voltage	sensor	in	wild	type	KCNQ2	channels,	and	this	would	underlie	a	change	in	voltage	sensor	equilibrium	in	the	presence	of	ICA73.	In	the	case	of	the	mutants	mentioned	above,	I	suggest	that	this	state-preference	is	 lost,	and	that	ICA73	can	bind	the	activated	and	resting	voltage-sensor	conformations	with	similar	energetics.	As	a	result,	the	voltage-dependence	of	channel	opening	should	be	barely	altered,	but	the	open	probability	of	channels	bound	by	 ICA73	 in	 the	 open	 state	 could	 still	 be	 increased,	 thereby	 potentiating	 overall	current	magnitude.	 Further	 experiments	 to	 test	 this	 hypothesis	 can	provide	more	insight	 on	 how	 ICA73	 and	 other	 similar	 compounds	 work.	 This	 developing	understanding	will	be	especially	important	because	in	drug	design,	state-dependent	interactions	of	drugs	with	their	targets	can	be	an	advantageous	property,	allowing	for	more	 specific	 targeting	 of	 hyperactive	 receptors	 (Tao	 et	 al.,	 2006).	 Thus,	 these			 75	experiments	will	hopefully	lay	a	valuable	foundation	for	further	understanding	of	the	mechanism	of	action	and	potential	therapeutic	uses	for	KCNQ	channel	activators.		Conclusion:		 This	thesis	has	revealed	novel	aspects	of	KCNQ	channel	activators	as	well	as	details	of	structural	and	functional	elements	of	KCNQ	channels.	Together,	the	findings	can	aid	in	the	improvement	of	drug	design	as	it	is	now	clear	that	there	are	multiple	mechanism	of	actions	of	compounds	acting	on	M-channels.	Importantly,	with	deeper	understanding	of	the	differences	between	KCNQ	subtypes,	the	aim	for	more	specific	therapeutic	targets	can	hopefully	be	achieved.	 			 76	References	Adams	PR,	Brown	DA	(1980)	Luteinizing	hormone-releasing	factor	and	muscarinic	agonists	 act	 on	 the	 same	 voltage-sensitive	 K+-current	 in	 bullfrog	 sympathetic	neurones.	Br	J	Pharmacol	68:353-355.	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