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Chan, Magnus

University of British Columbia

The cardiac delayed rectifier potassium current, IKs, formed by the co-assembly of KCNQ1 and KCNE1 subunits, plays a critical role in cardiac repolarization, acting as a reserve to adapt to higher heart rates and compensating when other repolarization currents are impaired, thereby preventing arrhythmias. Loss of function mutations in these subunits impair cardiac repolarization and are associated with inherited long QT syndrome, increasing the risk of developing a polymorphic ventricular tachycardia known as Torsades de Pointes, which can result in syncope and sudden death. Therapeutic interventions targeting the IKs channel directly to reverse the malignant effects of long QT syndrome hold significant potential but have historically been challenging due to the previously unknown binding sites for IKs activators. In this thesis, I delved into the binding mechanisms of two well-known IKs activators, mefenamic acid and 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS), and successfully elucidated their binding sites using electrophysiological and structural biology techniques. These activators potentiate the IKs current by inducing and stabilizing a conformational change in an otherwise hidden binding pocket within the S1/KCNE1/pore complex, slowing channel deactivation. Furthermore, I uncovered a dual mechanism of action for mefenamic acid on the IKs channel. At high concentrations (300 µM), mefenamic acid inhibits wild-type IKs current amplitude while preserving the delay in channel deactivation. Moreover, KCNE1 and KCNQ1 mutations in the previously elucidated binding pocket unmasked the inhibitory actions at lower drug concentrations. I propose that a lower affinity, secondary binding site near the primary binding site is responsible for inhibition, and specific mutations reveal the site. Finally, I demonstrated the graded stoichiometric effects of mefenamic acid on the wild-type IKs channel complex. The K41C mutation in KCNE1, which disrupts the primary binding site, abolishes the effects of mefenamic acid. My findings further quantified this graded loss of effect, showing that the response to mefenamic acid diminishes as the proportion of K41C-mutated subunits increases. Using mefenamic acid as an archetype for designing therapeutically useful IKs agonists, results from this thesis provide critical insights into the molecular mechanisms of IKs modulation and offers future directions for developing targeted therapies.

Text

2025-05-02

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/90959

Experimental Medicine

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/