Elsevier

Heart Rhythm

Volume 12, Issue 2, February 2015, Pages 386-394
Heart Rhythm

Microscopic mechanisms for long QT syndrome type 1 revealed by single-channel analysis of IKs with S3 domain mutations in KCNQ1

https://doi.org/10.1016/j.hrthm.2014.10.029Get rights and content

Background

The slowly activating delayed rectifier current IKs participates in cardiac repolarization, particularly at high heart rates, and mutations in this K+ channel complex underlie long QT syndrome (LQTS) types 1 and 5.

Objective

The purpose of this study was to determine biophysical mechanisms of LQT1 through single-channel kinetic analysis of IKs carrying LQT1 mutations in the S3 transmembrane region of the pore-forming subunit KCNQ1.

Methods

We analyzed cell-attached recordings from mammalian cells in which a single active KCNQ1 (wild type or mutant) and KCNE1 complex could be detected.

Results

The S3 mutants of KCNQ1 studied (D202H, I204F, V205M, and S209F), with the exception of S209F, all led to a reduction in channel activity through distinct kinetic mechanisms. D202H, I204F, and V205M showed decreased open probability (Po) compared with wild type (0.07, 0.04, and 0.12 vs 0.2); increased first latency from 1.66 to >2 seconds at +60 mV (I204F, V205M); variable-to-severe reductions in open dwell times (≥50% in V205M); stabilization of closed states (D202H); and an inability of channels to reach full conductance levels (V205M, I204F). S209F is a kinetic gain-of-function mutation with a high Po (0.40) and long open-state dwell times.

Conclusion

S3 mutations in KCNQ1 cause diverse kinetic defects in IKs, affecting opening and closing properties, and can account for LQT1 phenotypes.

Introduction

Coassembly of KCNQ1 and KCNE1 forms IKs,1 whose voltage- and time-dependent kinetics are critical to its function late in the cardiac action potential where it contributes to repolarization of the cardiomyocyte. Mutations in either KCNQ1 or KCNE1 lead to long QT syndrome (LQTS) types 1 and 5, and mutations in KCNQ1 can lead to short QT syndrome and atrial fibrillation.2

Our characterization of the wild-type (WT) IKs channel complex at the single-channel level has revealed infrequent openings with a low open probability (Po) <0.2 even at potentials of +60 mV, outside the physiologic range.3 The channels exhibit a long first latency to opening (mean 1.67 seconds at room temperature and +60 mV) and activate through several subconducting states before reaching a fully open level where they reside for approximately 50% of the time, flickering rapidly in nondeactivating bursts through to the end of the depolarization.3

These single-channel data suggest several mechanisms by which mutations not affecting channel trafficking, such as those in the lower KCNQ1 S3 domain,4 might impair IKs function. In this study, we subjected 4 S3 mutants in KCNQ1 (D202H, V205M, I204F, and S209F) to single-channel analysis to understand some microscopic kinetic mechanisms by which loss-of-function occurs in these mutants. We found each mutant defective in single or multiple kinetic ways, including changes in the latency to first opening, intraburst closings, premature termination of bursts, and stabilization of subconducting states.

Section snippets

Methods

Electrophysiologic studies were performed at 22oC on transiently transfected mouse ltk– cells prepared, transfected, and analyzed as previously described in detail.3, 4 Single-channel records were analyzed after digital filtering at 200 Hz. Capacitive currents were removed by subtracting the average of sweeps obtained at the same voltage that showed no channel activity. Half-amplitude threshold analysis5 and a rise time of 1.3 ms were used to detect events and generate idealized records from

Whole-cell properties of IKs LQTS mutants

The whole-cell electrophysiologic properties of several LQTS-causing mutations in the S3 helix of KCNQ1 are shown in Figure 1.4 The mutants D202H, I204F, and V205M all cause significant depolarizing shifts in the voltage dependence of opening (G-V) compared to WT (Figures 1A and 1C). Each mutation has unique effects on channel kinetics: D202H causes very rapid deactivation, I204F causes particularly slow activation, and V205M causes both slowed activation and fast deactivation (Figure 1B). In

Discussion

In this first single-channel study of LQT1 mutations, we uncovered multiple biophysical mechanisms that functionally result in the LQTS phenotype. These mechanisms range from the apparent stabilization of lower subconducting states in V205M and I204F to dramatic reductions in open probability (I204F) and large alterations in the energy barriers between open and closed states (D202H and V205M). These all represent previously unrecognized mechanisms by which loss of function in LQT1 can be

Summary

Single-channel recordings of IKs provide microscopic insight into the pathogenic mechanisms of LQTS caused by mutations in KCNQ1. We directly observed that D202H, I204F, and V205M destabilize open states with additional effects on closed states that differ between the D202H and I204F/V205M mutations. In addition, we extend our observations of subconductance states in IKs to mutations (V205M and I204F) that cause stabilization of lower subconductance behavior and present novel loss-of-function

References (14)

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This work was supported by the Heart and Stroke Foundation of British Columbia and Yukon and Canadian Institutes for Health Research grants to Dr. Fedida; and a Natural Sciences and Engineering Research Council of Canada scholarship to Mr. Daniel Werry.

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