ML277 regulates KCNQ1 single-channel amplitudes and kinetics, modified by voltage sensor state

Abstract

KCNQ1 is a pore-forming K+ channel subunit critically important to cardiac repolarization at high heart rates. (2R)-N-[4-(4-methoxyphenyl)-2-thiazolyl]-1-[(4-methylphenyl)sulfonyl]-2 piperidinecarboxamide, or ML277, is an activator of this channel that rescues function of pathophysiologically important mutant channel complexes in human induced pluripotent stem cell–derived cardiomyocytes, and that therefore may have therapeutic potential. Here we extend our understanding of ML277 actions through cell-attached single-channel recordings of wild-type and mutant KCNQ1 channels with voltage sensor domains fixed in resting, intermediate, and activated states. ML277 has profound effects on KCNQ1 single-channel kinetics, eliminating the flickering nature of the openings, converting them to discrete opening bursts, and increasing their amplitudes approximately threefold. KCNQ1 single-channel behavior after ML277 treatment most resembles IO state-locked channels (E160R/R231E) rather than AO state channels (E160R/R237E), suggesting that at least during ML277 treatment, KCNQ1 does not frequently visit the AO state. Introduction of KCNE1 subunits reduces the effectiveness of ML277, but some enhancement of single-channel openings is still observed.

Molecular simulations reveal a mechanism for enhanced allosteric coupling between voltage-sensor and pore domains in KCNQ1 explaining its activation by ML277

“…Third, MD simulations indicated that that ML277 modulates allosteric networks in KCNQ1 and increases the efficiency of allosteric signaling between the VSD and the PD. This result complements previous functional studies (11, 30, 31) showing that ML277 enhances VSD-PD coupling to increase KCNQ1 channel conductivity, specifically when the channel adopts the AO state. The MD simulations provided an atomic-level picture of the coupling process and a means to evaluate drug action.…”

“…Previous studies demonstrated that the activity of ML277 is dependent on the presence of KCNE1 (28–31). The ML277 effect was reduced with increasing expression levels of KCNE1.…”

“…Saturating KCNE1 levels led to little (29) or no (28) effect of ML277 on whole-cell currents and abolished the ML277-induced slowing of channel deactivation. On the other hand, at the single channel level, KCNQ1-KCNE1 channels were still potentiated by ML277 (31). Channel openings were more frequent and had larger amplitudes in presence of ML277, but were more flickery compared to openings of single KCNQ1 channels with ML277 (31).…”

“…We generated the G272V, F275L, S276A, F279L, C331G, A336G mutants that are located in the predicted ML277 binding site. These channels were transiently expressed in CHO cells stably expressing KCNE1 (CHO-E1 cells) and their consequences were determined by measuring ML277induced effects on peak and tail current density, voltage-dependence of activation V 1/2 and deactivation rate, three channel properties previously described as targets of ML277 modulation (28)(29)(30)(31).…”

“…ML277 potentiates the current amplitude of KCNQ1 in both whole-cell and single-channel configurations, hyperpolarizes the voltage-dependence of activation V 1/2 and slows channel deactivation (28–31). Similar to other KCNQ1 activators (e.g., R-L3 (20)), the activity of ML277 on KCNQ1-KCNE1 channels depends on the subunit stoichiometry (28, 31). The effect of ML277 on KCNQ1 channel current was found to be less with greater KCNE1 expression, with little (29) or no effect (28, 30) observed at saturating KCNE1 levels.…”