Mechanistic basis for LQT1 caused by S3 mutations in the KCNQ1 subunit of IKs

Abstract

Long QT interval syndrome (LQTS) type 1 (LQT1) has been reported to arise from mutations in the S3 domain of KCNQ1, but none of the seven S3 mutations in the literature have been characterized with respect to trafficking or biophysical deficiencies. Surface channel expression was studied using a proteinase K assay for KCNQ1 D202H/N, I204F/M, V205M, S209F, and V215M coexpressed with KCNE1 in mammalian cells. In each case, the majority of synthesized channel was found at the surface, but mutant IKs current density at +100 mV was reduced significantly for S209F, which showed ∼75% reduction over wild type (WT). All mutants except S209F showed positively shifted V1/2’s of activation and slowed channel activation compared with WT (V1/2 = +17.7 ± 2.4 mV and τactivation of 729 ms at +20 mV; n = 18). Deactivation was also accelerated in all mutants versus WT (126 ± 8 ms at −50 mV; n = 27), and these changes led to marked loss of repolarizing currents during action potential clamps at 2 and 4 Hz, except again S209F. KCNQ1 models localize these naturally occurring S3 mutants to the surface of the helices facing the other voltage sensor transmembrane domains and highlight inter-residue interactions involved in activation gating. V207M, currently classified as a polymorphism and facing lipid in the model, was indistinguishable from WT IKs. We conclude that S3 mutants of KCNQ1 cause LQTS predominantly through biophysical effects on the gating of IKs, but some mutants also show protein stability/trafficking defects, which explains why the kinetic gain-of-function mutation S209F causes LQT1.

Single-channel basis for the slow activation of the repolarizing cardiac potassium current, I Ks

“…This result suggested that 0.46 pA is about the lower limit for the single-channel current at this potential. However, we felt that a much better way to show directly that the single-channel amplitude at full opening is ∼0.5 pA at +60 mV was to make single-channel recordings from a gating mutant of I Ks that we previously have shown (25) to have a much higher Po than WT channels. The whole-cell findings from the S209F mutant are summarized in Fig.…”

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

“…The open dwell times were reduced to half that of WT ( Figure 4G and Table 1), consistent with the accelerated deactivation seen at the whole-cell level. 4 Closed dwell times ( Figure 4H) were best fit with 3 components, with only C f being different: 26.2 ms compared to 44.9 ms for WT (Table 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).…”

“…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.…”

“…4 This mutant is of interest because of its contrasting kinetic gain-of-function, with channels opening at more negative potentials and staying open longer upon repolarization. At þ60 mV, the channel cycles between periods of low and very high activity, giving an overall Po of 0.4, twice that of WT ( Figures 7A, 7D, and 7E).…”