Microscopic mechanisms for long QT syndrome type 1 revealed by single-channel analysis of IKs with S3 domain mutations in KCNQ1
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)
- et al.
Electrostatic interactions of S4 voltage sensor in Shaker K+ channel
Neuron
(1995) - et al.
A KCNQ1 V205M missense mutation causes a high rate of long QT syndrome in a First Nations community of northern British Columbia: a community-based approach to understanding the impact
Genet Med
(2008) - et al.
Coassembly of KvLQT1 and minK (IsK) proteins to form cardiac IKs potassium channel
Nature
(1996) - et al.
De novo KCNQ1 mutation responsible for atrial fibrillation and short QT syndrome in utero
Cardiovasc Res
(2005) - et al.
Single-channel basis for the slow activation of the repolarizing cardiac potassium current, I(Ks)
Proc Natl Acad Sci U S A
(2013) - et al.
Mechanistic basis for LQT1 caused by S3 mutations in the KCNQ1 subunit of I(Ks)
J Gen Physiol
(2010) - et al.
Fitting and statistical analysis of single-channel records
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The Genetics of Cardiovascular Disease in Canadian and International Aboriginal Populations
2015, Canadian Journal of CardiologyCitation Excerpt :A novel missense mutation, c.613 G>A (V205M) in the S3 transmembrane region in potassium channel, voltage gated KQT-like subfamily Q, member 1 (KCNQ1) (LQTS1), was identified in individuals with LQTS. Cellular mechanistic studies confirm the mutation significantly affects the function of Iks.72,168 In this population, associated sudden deaths (usually of young women, aged 25-50) and a prolonged QTc correlates with mutation carriers compared with relatives without the mutation, which supports a clinical predisposition to LQTS because of this mutation.72,73
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.