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T-type calcium channels functionally interact with spectrin (α/β) and ankyrin B Garcia-Caballero, Agustin; Zhang, Fang-Xiong; Hodgkinson, Victoria; Huang, Junting; Chen, Lina; Souza, Ivana A; Cain, Stuart; Kass, Jennifer; Alles, Sascha; Snutch, Terrance P; Zamponi, Gerald W May 2, 2018

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RESEARCH Open AccessT-type calcium channels functionallyinteract with spectrin (α/β) and ankyrin BAgustin Garcia-Caballero1, Fang-Xiong Zhang1, Victoria Hodgkinson1, Junting Huang1, Lina Chen1, Ivana A. Souza1,Stuart Cain2, Jennifer Kass2, Sascha Alles2, Terrance P. Snutch2 and Gerald W. Zamponi1*AbstractThis study describes the functional interaction between the Cav3.1 and Cav3.2 T-type calcium channels and cytoskeletalspectrin (α/β) and ankyrin B proteins. The interactions were identified utilizing a proteomic approach to identify proteinsthat interact with a conserved negatively charged cytosolic region present in the carboxy-terminus of T-type calciumchannels. Deletion of this stretch of amino acids decreased binding of Cav3.1 and Cav3.2 calcium channels tospectrin (α/β) and ankyrin B and notably also reduced T-type whole cell current densities in expression systems.Furthermore, fluorescence recovery after photobleaching analysis of mutant channels lacking the proximal C-terminusregion revealed reduced recovery of both Cav3.1 and Cav3.2 mutant channels in hippocampal neurons. Knockdown ofspectrin α and ankyrin B decreased the density of endogenous Cav3.2 in hippocampal neurons. These findings revealspectrin (α/β) / ankyrin B cytoskeletal and signaling proteins as key regulators of T-type calcium channels expressed in thenervous system.Keywords: T-type channels, Spectrin (α/β), Ankyrin B, Trafficking, Cav3.1, Cav3.2IntroductionT-type calcium channels are important regulators ofneuronal excitability and low threshold-mediated exocyt-osis [1, 2]. The mammalian genome encodes three differentT-type calcium channels (Cav3.1, Cav3.2 and Cav3.3) [3].The pore forming Cavα1 subunits of these channels arecomprised of four homologous transmembrane domainslinked by cytoplasmic segments and flanked by intracellu-lar N- and C-terminus structures. T-type channels arehighly expressed in the central nervous system includingneocortex, cerebellum, thalamus and hippocampus [4] andhave been linked to pathophysiologies such as idiopathicgeneralized epilepsies [5] and tremor [6]. The Cav3.2 sub-type is expressed in dorsal root ganglion neurons andspinal cord where its dysregulation contributes to the de-velopment of inflammatory and neuropathic pain [7]. Not-ably, T-type calcium channel blockers are effective inattenuating absence seizures and chronic pain in rodentmodels [8, 9] and have also been shown efficacious in ahuman model of inflammatory pain. In this context, under-standing how these channels are regulated and traffickedto the cell surface is of importance.T-type channels are known to interact with numerousregulatory proteins including CamKII [10], G-proteins[11], calcineurin [12], calmodulin [13], syntaxin [1] andcalnexin [14], and to form protein complexes with mem-bers of the potassium channel family such as Kv4, KCa3.1, and KCa1.1 [15–17]. Many of these interactions ap-pear to involve the cytosolic carboxy-terminal region ofCav3.x proteins and here we utilized a proteomic ap-proach to identify additional interacting partners. In par-ticular, we focused on a stretch of conserved negativelycharged residues located in the proximal carboxy-terminal regions of Cav3.1 and Cav3.2 channels. Massspectrometry identified spectrins as Cav3.2 C-terminalinteracting proteins. We further describe the interactionsand functional regulation of Cav3.1 and Cav3.2 channelswith spectrin (α/β) and ankyrin B in both exogenousexpression and native systems, revealing that these inter-actions regulate whole cell current density. Together, theresults indicate that cytoskeletal elements are importantregulators of T-type calcium channel function andphysiology.* Correspondence: zamponi@ucalgary.ca1Department of Physiology and Pharmacology, Hotchkiss Brain Institute andAlberta Children’s Hospital Research Institute, Cumming School of Medicine,University of Calgary, 3330 Hospital Dr. NW, Calgary T2N 4N1, CanadaFull list of author information is available at the end of the article© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Garcia-Caballero et al. Molecular Brain  (2018) 11:24 https://doi.org/10.1186/s13041-018-0368-5ResultsSpectrins interact with T-type calcium channelsThe proximal C-terminus regions of Cav3.1 and Cav3.2contain a conserved α-helical (http://bioinf.cs.ucl.ac.uk/psipred) stretch of charged amino acid residues (aminoacids 1851–1875 in Cav3.1, and 1860–1884 in Cav3.2)(Fig. 1a). To determine whether this motif is a bindingregion for regulatory proteins, we used a proteomicapproach with a corresponding Cav3.2 CT synthetic pep-tide conjugated with biotin as bait for interacting pro-teins in mouse brain lysates. Bound proteins wereresolved in a denaturing Coomassie gel (Fig. 1b) andprotein bands that appeared in samples incubated withthe Cav3.2CT bait, but not in samples incubated withthe control scramble peptide, were excised and analyzedby MALDI/TOF mass spectrometry. This analysisyielded hits for three cytoskeletal proteins: spectrin- αII(SPTAN1) and two isoforms of spectrin-β (SPTBN1 andSPTBN2) (Fig. 1c), with the former showing the highestscore. Spectrin is a heterodimeric protein comprised ofα- and β- subunits (280 and 246 kDa, respectively) thatform a supercoiled triple-helix structure through 106residue modules known as “spectrin repeats” [18]. Thisparticular structural organization allows spectrin to ex-pand and contract to remodel the cytoskeleton and,hence, cellular architecture [19]. In humans, there aretwo α and five β spectrin subunit genes. Spectrin-αI isexpressed in erythroid cells whereas spectrin-αII isexpressed in all nonerythroid cells, including the brainwhere it is important for synaptic transmission [20] andparticipates in neurotransmitter release [21]. Spectrinspossess multiple interacting domains such as a pleckstrinhomology domain, a Src homology 3 domain (SH3), a cal-cium binding EF hand domain, an ankyrin binding repeatand an actin binding domain, that interact with a diverseset of cell signaling proteins, receptors and ion channels[18]. To confirm SPTAN1-Cav3 interactions, we per-formed co-immunoprecipitations between SPTAN1 andeither Cav3.1 or Cav3.2. Figure 1d and e show thatSPTAN1 co-immunoprecipitated with both T-type chan-nel isoforms from mouse brain lysates.We next asked whether deletion of the helical regionin the proximal C-terminus regions of Cav3.1 and Cav3.2 could alter their association with SPTAN1. For thispurpose, we expressed GFP-tagged wild type ordeletion-mutant channels in tsA-201 cells and per-formed co-immunoprecipitations between the exogen-ously expressed channels and endogenous spectrin.Consistent with the data in Fig. 1, both GFP-taggedCav3.1 and Cav3.2 channels co-immunoprecipitatedwith SPTAN1 (Fig. 2a and b). Deleting the helical region(Cav3.1-GFP ΔCT (1851–1875), Cav3.2-GFP ΔCTacdbeFig. 1 Identification of spectrin (a/β) as a Cav3.1 / Cav3.2 calcium channel interacting protein. (a) Conserved proximal C-terminus region of Cav3.1and Cav3.2 calcium channels. (b) Proteins bound to scramble (lane 1) or Cav3.2 CT 1860–1884 biotinylated peptides (lane 2) from mouse wholebrain lysates, as seen by Coomassie staining (c) Mass spectrometry analysis of proteins bound to the Cav3.2 CT 1860–1884 biotinylated peptideby affinity precipitation assay using mouse whole brain lysates. (d) Cav3.1 or (e) Cav3.2 immunoprecipitates from mouse whole brain lysates wereprobed for spectrin αII (SPTAN1) by Western blot. An actin loading control is shownGarcia-Caballero et al. Molecular Brain  (2018) 11:24 Page 2 of 10(1860–1884)) reduced, but did not completely eliminatebinding of SPTAN1 to both channel isoforms (Fig. 2aand b), confirming that this region is involved in spec-trin interactions, but also that spectrin may associatewith contact sites in other regions of the channels. Theseputative additional interactions do not appear to involvethe distal C-terminus regions, since deletion of residues1875–2377 in Cav3.1 (Cav3.1-GFP ΔCT (1875–2377))had no effect on co-immunoprecipitations with SPTAN1(Additional file 1: Figure S1).Given that other spectrin isoforms were pulled downwith the C-terminal helix bait, we wanted to confirmthese interactions in tsA-201 cells. Unfortunately, thesecells do not appear to express spectrin-β1, and hence wecould not test these interactions with this expressionsystem. However, tsA-201 cells do express spectrin-βII(SPTBN2) which could be co-immunoprecipitated withboth Cav3.1-GFP and Cav3.2-GFP (Fig. 2c and d). Aswith SPTAN1, the SPTBN2 interactions were signifi-cantly weakened by deleting the helical regions in theCav3.1 and Cav3.2 C-termini (Fig. 2c and d). As thesedata are based upon co-immunoprecipitations and thatSPTAN1 and SPTBN2 interact with each other, it is un-clear if either the two spectrin isoforms is the primaryinteraction partner, or whether there may be anotherintermediate protein(s) involved in linking the channelsto the cytoskeleton. Taken together, these data indicatethat spectrins physically interact with a putative helicalregion conserved in the C-terminal domains of Cav3.1and Cav3.2 calcium channels.Ankyrin B interacts with T-type calcium channelsDirect interactions with spectrin have been reported forepithelial sodium channels, whereas neuronal voltage-gated sodium channels are reportedly linked to spectrinvia ankyrin B or G [22]. We therefore examined bindingof ankyrin B to Cav3.1 and Cav3.2 channels. Followingexpression of Cav3.1-GFP and Cav3.2-GFP constructs intsA-201 cells immunoprecipitates with GFP were probedwith an ankyrin B antibody, revealing interactions withboth channel isoforms (Fig. 3a and b). Deletion of theproximal C-terminal alpha helix in the two channels re-duced ankyrin B interactions with Cav3.1 and to a lesserextent with Cav3.2 (Fig. 3a and b). These data raise thepossibility that, similar to voltage gated sodium chan-nels, spectrin may link to Cav3 channels via ankyrininteractions.a bc dFig. 2 Effect of Cav3.1-GFP and Cav3.2-GFP C-terminal deletions on SPTAN1 and SPTBN2 binding in tsA-201 cells. (a) Cav3.1-GFP ΔCT (1851–1875)and wild type channel immunoprecipitates probed with anti-Spectrin αII (SPTAN1) polyclonal antibody. Densitometry analysis of SPTAN1 boundto Cav3.1-GFP immunoprecipitates is shown. (P = 0.0051, n = 3). (b) Cav3.2-GFP ΔCT (1860–1884) and wild type channel immunoprecipitatesprobed with anti-Spectrin αII (SPTAN1) polyclonal antibody. Densitometry analysis of SPTAN1 bound to Cav3.2-GFP immunoprecipitates is shown(P = 0.0005, n = 3). (c) Cav3.1-GFP ΔCT (1851–1875) and wild type channel immunoprecipitates probed with anti-Spectrin βII (SPTBN2)polyclonal antibody. Densitometry analysis of SPTBN2 bound to Cav3.1-GFP immunoprecipitates is shown (P = 0.0085, n = 3). (d) Cav3.2-GFP ΔCT(1860–1884) and wild type channel immunoprecipitates probed with anti-Spectrin βII (SPTBN2) polyclonal antibody. Densitometry analysis of SPTBN2bound to Cav3.2-GFP immunoprecipitates is shown. (P= 0.0047, n = 3)Garcia-Caballero et al. Molecular Brain  (2018) 11:24 Page 3 of 10Functional role of the Cav3 cytoskeletal interactingdomainWe next examined whole cell calcium currents fromwild type and deletion-mutant Cav3.1 expressed intsA-201 cells by recording current-voltage relation-ships for wildtype Cav3.1 (wt) and mutant Cav3.1(Δ1851–1875). Deletion of (MKHLEESNKEAKEEAE-LEAELELE) reduced Cav3.1 current density by ap-proximately 75% (wt Cav3.1 = − 238.68 ± 20.36 pA/pFat − 20 mV; Cav3.1 Δ1851–1875 = − 60.96 ± 12.71 pA/pFat − 20 mV; P < 0.001, n = 10); Fig. 4a and b). Similarly, forwt Cav3.2 and mutant Cav3.2 (Δ1860–1884) deletion ofconserved sequence (MKHLEESNKEAREDAELDAEIELE)significantly reduced the current density of Cav3.2(wt = − 95.84 ± 4.45 pA/pF at − 20 mV; Cav3.2 Δ1860–1884 = − 35.67 ± 4.60 pA/pF at − 20 mV; P < 0.001, n =10; Fig. 4c and d). There were no changes in thevoltage-dependence of activation of the channels, norwere there any changes in channel kinetics (notshown), suggesting that the reduced current densitiesmight be due to fewer channels in the plasma mem-brane. This notion is supported by experiments exam-ining the cell surface pool of transiently expressedCav3.1 channels incubated with a cell permeant (i.e.,Tat epitope fused) disruptor peptide corresponding tothe putative spectrin interaction site. When comparedto a scrambled Tat control peptide, Cav3.1 cell sur-face expression was diminished by the disruptor pep-tide (Additional file 1: Figure S2), indicating that thecytoskeletal interactions may regulate the cell surfacedensity of Cav3.1 channels.Next, we tested whether deletion of the cytoskeletoninteraction region in Cav3 channels altered Cav3.x chan-nel trafficking. To address this, we transfected full-length and deletion-mutant Cav3-GFP channels intocultured hippocampal neurons and performed fluores-cence recovery after photobleaching (FRAP) experiments.Fluorescence intensity mediated by the full-length Cav3.1-GFP and Cav3.2-GFP channels was found to recover morestrongly than signals mediated by the deletion mutants(Fig. 5a-c), suggesting that the reduced ability of the dele-tion mutants to interact with cytoskeletal proteins affectsthe lateral mobility or insertion of new Cav3 channels inthe plasma membrane.We then examined the expression of endogenous Cav3channels in cultured hippocampal neurons in responseto knockdown of SPTAN1 and ankyrin B. For these ex-periments we focused on the Cav3.2 isoform. Western blotsabFig. 3 Effect of Cav3.1-GFP and Cav3.2-GFP C-terminal deletions on ankyrin B binding in tsA-201 cells. (a) Cav3.1-GFP ΔCT (1851–1875) and wildtype channel immunoprecipitates probed with anti-ankyrin B polyclonal antibody. Densitometry analysis of ankyrin B bound to Cav3.1-GFPimmunoprecipitates is shown (P = 0.0005, n = 4). (b) Cav3.2-GFP ΔCT (1860–1884) and wild type channel immunoprecipitates probed with anti-ankyrinB polyclonal antibody. Densitometry analysis of ankyrin B bound to Cav3.2-GFP immunoprecipitates is shown (P = 0.015, n = 4)Garcia-Caballero et al. Molecular Brain  (2018) 11:24 Page 4 of 10confirmed the expression and subsequent shRNA-mediatedknockdown of endogenous SPTAN1 and ankyrin B in hip-pocampal neurons (Fig. 6a and d). We performed immuno-staining experiments in which endogenous Cav3.2 channelswere stained with a Cav3.2 antibody. Cav3.2 channel ex-pression was evident in cell bodies, axons and proximaldendrites (Fig. 6b and e). Upon shRNA-mediated knock-down of SPTAN1, total Cav3.2 fluorescence was consist-ently diminished (Fig. 6b and c). A similar effect wasobserved upon knockdown of ankyrin B (Fig. 6e and f).Altogether, these data are consistent with the reducedchannel current densities and FRAP signals observed upondepletion of the Cav3 interaction domain, and support aneffect of cytoskeletal elements in the expression of native T-type calcium channels.DiscussionIn this study, we used a proteomic approach to identifyinteracting partners of the proximal Cav3.1 and Cav3.2C-terminus regions. The screen identified both α and βspectrin as both interacting partner with the first 24amino acids of the C terminal region. Both spectrinsubtypes are membrane cytoskeleton components [23,24] and there is a growing body of literature implicat-ing spectrins in the clustering of ion channels (e.g.,sodium and potassium channels) at specific subcellu-lar loci [25–27], often indirectly via specific ankyrins[22, 28, 29]. We confirmed the spectrin interactions viaco-immunoprecipitation from mouse brain tissue and alsoidentified ankyrin B as part of the putative Cav3.2/spectrinbinding complex. Deletion of 24 amino acid residues ofthe proximal Cav3 C-terminus from the full-length chan-nels reduced whole cell current densities in transient ex-pression systems. Furthermore, shRNA depletion of bothspectrin α and ankyrin B in hippocampal neurons reducedCav3.2 type channel expression. Finally, fluorescence re-covery experiments using GFP-tagged Cav3 channels inhippocampal neurons revealed that Cav3.1 and Cav3.2channels lacking the interaction domain display signifi-cantly reduced mobility in the plasma membrane and/ortrafficking to the membrane compared to wild type chan-nels. Taken together, we conclude that cytoskeleton inter-actions are important determinants of T-type calciumchannel trafficking to and within the plasma membrane.ca bdFig. 4 Calcium currents evoked by wild type and mutant Cav3.1 and Cav3.2 channels in tsA-201 cells. (a) Current-voltage (I/V) relationship of wildtype or mutant Cav3.1-GFP ΔCT (1851–1875) mutant channels (n = 10). Values are represented as means +/− S.E. The solid lines are fits with theBoltzmann equation. (b) Mean peak current density representation from wild type or mutant Cav3.1-GFP ΔCT (1851–1875) channels (P < 0.001,n = 10). Asterisks denote statistical significance relative to wild type (****P< 0.0001, Student’s t-test). (c) Current-voltage (I/V) relationship of wild type ormutant Cav3.2-GFP ΔCT (1860–1884) channels (n= 18–19). Values are represented as means +/− S.E. The solid lines are fits with the Boltzmann equation.(d) Mean peak current density representation from wild type or mutant Cav3.2-GFP ΔCT (1860–1884) channels (P < 0.001, n = 10). Asterisks denotestatistical significance relative to wild type (****P < 0.0001, Student’s t-test)Garcia-Caballero et al. Molecular Brain  (2018) 11:24 Page 5 of 10The molecular mechanisms by which these cytoskeletalinteractions affect T-type channel trafficking remain to bedetermined. Within the plasma membrane, interactions ofthe channel with the cytoskeleton may facilitate the lateraltrafficking due to dynamic cytoskeletal rearrangements. Itis also possible that interactions with spectrin facilitate theeffective translocation of the channel from the endoplas-mic reticulum to the cell surface, either by facilitatingtransport, or by occluding an ER retention signal.The data indicate that the proximal carboxy-terminusregions of Cav3.1 and Cav3.2 binds to spectrins (α/β) viaa 24 amino acid stretch containing many negativelycharged residues located in close proximity to the trans-membrane domain of these channels. This interactionmay be responsible for promoting binding of ankyrin Bto the complex, likely via direct binding to the describedα/β spectrin ankyrin binding repeat [18]. In contrastwith the previously identified proline rich motif presentin other ion channels [30] that binds to the SH3 domainin spectrins, this novel motif conserved across T-typecalcium channels is enriched with glutamic and asparticacids, possibly involved in electrostatic interactions withspectrin (α/β).Literature from the voltage-gated sodium channel fieldidentifies ankyrin as a key determinant of sodiumchannel clustering at Nodes of Ranvier [31, 32]. It is thuspossible that spectrin/ankyrin interactions with Cav3 me-diate a similar clustering/targeting role for localization ofT-type channels at specific subcellular loci. The observa-tion that deletion of the putative spectrin/ankyrin inter-action motif in both Cav3.1 and Cav3.2 channels greatlyimpeded fluorescence recovery in FRAP experiments andreduced whole cell current densities is consistent with arole of the cytoskeleton trafficking to and within theplasma membrane. Whether these interactions are in-volved in targeting the channels to specific membranecompartments such as nodal regions, or synaptic/den-dritic sites remains to be determined.In the nervous system, T-type calcium channels fulfilltwo major roles. One is to regulate the excitability ofneurons, being of particular importance in the corti-cothalamic circuitry wherein T-type channels are knownto mediate rebound bursting [33] and is of particularrelevance to the genesis of absence seizures [34]. A sec-ond major role that has emerged more recently is theircontribution towards low threshold-mediated neuro-transmitter release [1]. Indeed, Cav3 channels interactwith syntaxin 1A and have been shown to contribute tosynaptic release in dorsal horn synapses [7, 35, 36].This is of particular relevance for conditions such asab cFig. 5 Mobility of wild type and mutant Cav3.1 and Cav3.2 channels in hippocampal neurons. (a) Fluorescence recovery after photobleaching(FRAP) assay of Cav3.1-GFP wild type and ΔCT (1851–1875) mutant channels or Cav3.2-GFP wild type and ΔCT (1860–1884) mutant channelstransfected into mouse hippocampal neuron cultures. (b) Recovery (Fluorescence-Fluorescence bleach) of Cav3.1-GFP wild type and ΔCT (1851–1875)mutant channels. Recovery values are calculated as F(maximum after photobleach)-F(photobleach) (n = 8 WT, n = 8 mutant, P < 0.01, Student’s t-test).(c) Recovery (Fluorescence-Fluorescence bleach) of Cav3.2-GFP wild type and ΔCT (1860–1884) mutant channels. Recovery values are calculated asF(maximum after photobleach)-F(photobleach) (n = 11 WT, n = 12 mutant, P < 0.01, Student’s t-test)Garcia-Caballero et al. Molecular Brain  (2018) 11:24 Page 6 of 10neuropathic pain where increases in Cav3 channelcurrent density facilitate neuronal firing and synapticcommunications in the afferent pain pathway. Giventheir potent effects on T-type channel expression, spec-trins and ankyrins may act as regulatory elements forcontrolling nervous system function via T-type channelinteractions.The regulation of T-type calcium channels by cyto-skeletal elements is likely to extend beyond thenervous system. For example, ankyrin B is highlyexpressed in cardiac myocytes where it regulatesexcitation-contraction coupling in concert with othersignaling molecules [37]. Myocytes also express Cav3.1and Cav3.2 T-type calcium channels, especially duringearly post-natal development [38]. T-type channel expres-sion is however increased under pathological conditionssuch as cardiac hypertrophy [39] thus it would be interest-ing to determine whether T-type channel expressionincreases are related to cytoskeletal remodeling.ConclusionIn summary, we have identified cytoskeletal interactionsas a molecular mechanism for regulating T-type channelmobility and current density. It is possible that targetingthese interactions may offer a means for affecting T-typecalcium channel activity in disorders such as chronicpain and epilepsy.MethodsDrugs and peptidesHuman biotin-Cav3.2-CT 1860–1884, biotin-Cav3.2-CTscramble peptides (Genemed synthesis, San Antonio,Tx). SPTAN1 and ankyrin B shRNAs were purchasedfrom Thermo Scientific, Open Biosystems.Cell culture and transfectionHuman embryonic kidney tsA-201 cells were cultured asdescribed [40]. Cells were transfected with Lipofectaminea b cd e fFig. 6 SPTAN1 and ankyrin B knockdown effect on endogenous Cav3.2 calcium channel density in mouse and rat hippocampal neurons. (a)SPTAN1 expression levels in mouse hippocampal neurons treated with shRNA as seen by western blot. (b) Mouse hippocampal neurons untreated ortreated with shRNA for SPTAN1 were stained for Cav3.2 channels with a specific anti-Cav3.2 polyclonal antibody. (c) Cav3.2 channel intensity valuesfrom untreated neurons or neurons treated with SPTAN1 shRNA. (d) Ankyrin B expression levels in rat hippocampal neurons treated with shRNA asseen by western blot. (e) Rat hippocampal neurons untreated or treated with shRNA for ankyrin B were stained for Cav3.2 channels with a specificanti-Cav3.2 polyclonal antibody. (f) Cav3.2 channel intensity values from untreated neurons or neurons treated with specific ankyrin B shRNAGarcia-Caballero et al. Molecular Brain  (2018) 11:24 Page 7 of 102000 and used for biochemical and electrophysiologicalanalysis 48–72 h post-transfection.Hippocampal neuron primary culturesMouse or rat hippocampal neurons were dissociated asdescribed before [41] and seeded at low density ontocoverslips pretreated with poly-D-lysine (Sigma)followed by Laminin (Sigma) in 24-well plates. At daysix of culture, transfection of cDNA was performedusing Lipofectamine 2000 (Invitrogen) following themanufacturer’s instructions. We used 1.5 μg of cDNAper well with 2 μl of Lipofectamine. cDNA and Lipofec-tamine solution were mixed together for 30 min at roomtemperature. Cells were incubated in cDNA–Lipofecta-mine 2000 complexes for 2 h at 37 °C and coverslipswere placed back in their medium. Four days after trans-fection, immunostaining with GFP antibody (1500) wasconducted at 37 °C.PlasmidsTo generate the GFP-tagged Cav3.1 or Cav3.2, the cod-ing sequences of human Cav3.1 or human Cav3.2 wascloned into the pcDNA3.1(+) vector (Invitrogen) withstop codon removed; GFP was amplified by PCR andinserted into the C-terminus of Cav3.1 or Cav3.2. Todelete the specific fragment amino acids 1860–1884from Cav3.1 or Cav3.2, we used two-step PCR. The firstround of PCR amplified the upstream and downstreamflanking regions of that fragment. Products were purifiedand used as templates for the second round of PCR,which recombined these two regions together. The finalPCR product was used to replace wide type Cav3.1 orCav3.2 by making use of appropriate restriction sites inthe two flanking regions.Affinity precipitation of Cav3.2 interacting proteinsMouse brain proteins were solubilized in buffer (in mM;50 Tris pH 7.6, 150 NaCl, 1% Triton X-100, 1% NP40,10 EDTA, 10 EGTA and protease inhibitors). Solubleproteins were collected by centrifugation at 16,100 g for10 min. Supernatant fractions (500 μg) were preclearedby incubation with neutravidin beads for 1 h at 4 °C(Thermo Scientific) and then incubated in a modifiedsolubilization buffer (in mM; 50 Tris pH 7.6, 150 NaCl,0.2% Triton X-100, 0.2% NP40, 10 EDTA, 10 EGTA andprotease inhibitors) for 2 h at 4 °C with a human Cav3.2-carboxy-terminal (1860–1884 a.a.(MKHLEESNKEAREDAELDAEIELEM)) or a scramble (SEMADLEKAENHMDEIMEKAEEREL) biotinylated peptides (5 μg) cova-lently linked to a C-terminal biotin (Genemed synthesisInc., San Antonio, Tx). After Cav3.2-interacting proteinswere collected, samples were washed three times withmodified solubilization buffer. Bound proteins were ana-lyzed by SDS-PAGE and visualized by Coomassie bluestaining (Sigma). Visible bands were excised and samplesanalyzed by MALDI/TOF-MS (Bruker Instruments Co.,Bremen, Germany).Western blottingWestern blot analysis was performed using anti-actinmouse (Sigma), anti-GFP (Abcam) anti-αII-spectrin(Santa Cruz, Biotechnology, Inc.) and anti-ankyrin B(Santa Cruz Biotechnology, Inc.) rabbit antibodies. West-ern blot quantification was performed using densitom-etry analysis (Quantity One-BioRad software). Student’st-tests for unpaired data were performed to determinestatistical significance.Co-immunoprecipitation assaysMouse whole brain tissue or tsA-201 cells were lysed ina modified RIPA buffer (in mM; 50 Tris, 100 NaCl, 0.2%(v/v) Triton X-100, 0.2% (v/v) NP-40, 10 EDTA + prote-ase inhibitor cocktail, pH 7.5) that was used to co-immunoprecipitate Cav3.2 channels with spectrin (α/β)or ankyrin B proteins. Lysates were prepared by sonicat-ing samples at 60% pulse for 10 s and by centrifugationat 13,000 rpm for 15 min at 4 °C. Supernatants weretransferred to new tubes and solubilized proteins wereincubated with 50 μl of Protein G/A beads (Piercenet)and 2 μg of anti-Cav3.2 (H-300, Santa Cruz Biotechnolo-gies, Inc) antibody or anti-GFP antibody (Abcam) over-night while tumbling at 4 °C. Total inputs were takenfrom whole cell samples representing 4% of total proteinand probed for actin. Co-immunoprecipitates werewashed twice with (mM) 150 NaCl 50 Tris pH 7.5 buf-fer, beads were aspirated to dryness. Laemmli buffer wasadded and samples were incubated at 96 °C for 7 min.Eluted samples were loaded on 7.5% Tris-glycine gel andresolved using SDS-PAGE. Samples were transferred to0.45 mm polyvinylidenedifluoride (PDVF) membranesby dry transfer using an Iblot machine (Invitrogen).Electrophysiological recordingsWhole-cell voltage-clamp recordings for tsA-201 cellswere performed 72 h after transfection with cDNA(Cav3.1-GFP wild type (2 μg) or Cav3.1-GFP Mutant(2 μg) and Cav3.2-GFP wild type or Cav3.2-GFP Mutant(2 μg)). Recordings were conducted with 10 mM bariumas the charge carrier, using internal and external record-ing solutions; (in mM): 110 CsCl, 3 Mg-ATP, 0.5 Na-GTP, 2.5 MgCl2, 5 D-glucose, 10 EGTA, 10 HEPES(pH 7.3 with CsOH). The external solution contained (inmM): 10 BaCl2, 1 MgCl2, 140 TEACl, 10 D-glucose, 10HEPES (pH 7.2 with TEAOH). Currents were elicitedfrom a holding potential of − 100 mV and depolarizedfrom − 70 to + 50 mV with 10-mV increments. Data werecollected from multiple batches of transfections with simi-lar numbers of cells tested from the different groups. ForGarcia-Caballero et al. Molecular Brain  (2018) 11:24 Page 8 of 10data analysis, peak currents and cell capacitance weremeasured and converted into current density.FRAP assaysPrimary hippocampal neurons were transfected at 10DIV and imaged 48 h afterward. Transfections weredone with 2 μg of DNA/ matek dish with 6ul of Lipofec-tamine 2000 for 2 h.Photobleaching experiments: FRAP experiments wereimaged using a ZEISS 510 LSM. A ROI was selected onan axon proximal to the cell body of a neuron. After 3baseline images, 10 iterations of 100% laser power wasused to bleach the ROI, and recovery imaged for 13 min,with a picture every 5 s. Fluorescence intensity values werenormalized to an initial intensity of 100%. Cells wereexcluded if photobleaching did not decrease fluorescenceintensity to more than 50% of initial 100%. Traces: Fluor-escence intensity values are normalized to an initial valueof 100%. Intensity values were normalized to an initialvalue of 100%. Recovery values are calculated as F max-imum after photobleach- F photobleach.ImmunofluorescenceBriefly, cultured rat hippocampal neurons were washedtwice with PBS containing (mM) 1 MgCl2 and 2 CaCl2.Neurons were fixed with 4% paraformaldehyde for20 min at room temperature (RT) and washed 3 timesafter fixation. Then neurons were permeabilized withPBS containing 0.1% triton x-100 and 2 mg/ml BSA for30 min. Neurons were blocked with PBS containing 4%milk and 2 mg/ml BSA for 2 h at RT. Cav3.2 (mouseanti-Cav3.2, 1 μg/ml, Novus Biologicals,CA) primaryantibodies were incubated overnight at 4 °C. After wash-ing three times, the secondary antibodies Alexafluor 546conjugated Donkey anti-mouse (Thermo Fisher Scien-tific, CA) were incubated 2 h. All images were digitallycaptured with an 8 bit camera, thus giving grey level (in-tensity) values of 0–255. Immunostaining was visualizedusing a 40 × 0.4 NA objective lens on a Zeiss LSM 510META confocal systems, running Velocity 6.Data analysis and statisticsFor biochemical and electrophysiological analyses, datavalues are presented as mean ± SEM for n experiments.Statistical significance was determined using Student’s t testunless stated otherwise: *p < 0.05; ** p < 0.01; *** p < 0.001;NS, statistically not different.Additional fileAdditional file 1: Figure S1. Binding of SPTAN1 to Cav3.1-GFP ΔCT1875–2377 mutant channels lacking a distal C-terminus region. Cav3.1-GFPΔCT (1875–2377) and wild type channel immunoprecipitates fromtransfected tsA-201 cells probed with anti-Spectrin αII (SPTAN1)polyclonal antibody. Densitometry analysis of SPTAN1 bound toCav3.1-GFP immunoprecipitates is shown. Figure S2. DisruptingCav3.1 SPTAN1 interactions reduces cell surface expression of Cav3.1.Left: Surface biotinylation experiments on Cav3.1 channels transientlyexpressed in tsA-201 cells in the presence of a cell permeant Tatpeptide corresponding to the putative spectrin interaction site(Tat-Cav3.1-CT) on the channel, or a scrambled peptide sequence.Right: Densitometry analysis of Cav3.1 surface pool normalized to theactin control. Note that the Tat- Cav3.1-CT peptide reduces the cellsurface expression of the channel by ~ 40%. (DOC 118 kb)AbbreviationsFRAP: Fluorescence recovery after photobleaching; GFP: Green fluorescentprotein; PCR: Polymerase chain reaction; shRNA: Short hairpin ribonucleicacid, wt: wild typeFundingThis work was supported by a Discovery grant to GWZ from the NaturalSciences and Engineering Research Council. GWZ is a Canada Research Chairin Molecular Neuroscience. Work in the laboratory of TPS is supported by anoperating grant from the Canadian Institutes of Health Research (#10677)and the Canada Research Chair in Biotechnology and Genomics-Neurobiology.Availability of data and materialsAll data generated or analysed during this study are included in this publishedarticle [and its supplementary information files].Authors’ contributionsAG-C-performed biochemistry experiments, and with GWZ designed the studyand co-wrote the manuscript, FXZ, VH, IAS, SC, SA, JK and JH performedexperiments, LC harvested and cultured tissue, TS and GWZ supervisedexperiments, and GZ and TPS edited the manuscript. All authors read andapproved the final manuscript.Ethics approval and consent to participateProtocols for tissue harvesting from mice were approved by the University ofCalgary’s Animal Care Committee.Competing interestsThe authors declare that they have no competing interests.Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims in publishedmaps and institutional affiliations.Author details1Department of Physiology and Pharmacology, Hotchkiss Brain Institute andAlberta Children’s Hospital Research Institute, Cumming School of Medicine,University of Calgary, 3330 Hospital Dr. NW, Calgary T2N 4N1, Canada.2Michael Smith Laboratories and Djavad Mowafaghian Centre for BrainHealth, University of British Colombia, Vancouver, BC, Canada.Received: 7 March 2018 Accepted: 23 April 2018References1. Weiss N, Hameed S, Fernandez-Fernandez JM, et al. A ca(v)3.2/syntaxin-1Asignaling complex controls T-type channel activity and low-thresholdexocytosis. 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