Dysfunctional potassium channel subunit interaction as a novel mechanism of long QT syndrome.

BACKGROUND: The slowly-activating delayed rectifier current IKs contributes to repolarization of the cardiac action potential, and is composed of a pore-forming α-subunit, KCNQ1, and a modulatory ß-subunit, KCNE1. Mutations in either subunit can cause long QT syndrome, a potentially fatal arrhythmic disorder. How KCNE1 exerts its extensive control over the kinetics of IKs remains unresolved OBJECTIVE: To evaluate the impact of a novel KCNQ1 mutation on IKs channel gating and kinetics METHODS: KCNQ1 mutations were expressed in Xenopus oocytes in the presence and absence of KCNE1. Voltage clamping and MODELLER software were used to characterize and model channel function. Mutant and wt genes were cloned into FLAG, Myc and HA expression vectors to achieve differential epitope tagging, and expressed in HEK293 cells for immunohistochemical localization and surface biotinylation assay. RESULTS: We identified 2 adjacent mutations, S338F and F339S, in the KCNQ1 S6 domain in unrelated probands. The novel KCNQ1 S338F mutation segregated with prolonged QT interval and torsade de pointes; the second variant, F339S, was associated with fetal bradycardia and prolonged QT interval, but no other clinical events. S338F channels expressed in Xenopus oocytes had slightly increased peak conductance relative to wild type, with a more positive activation voltage. F339S channels conducted minimal current. Unexpectedly, S338F currents were abolished by co-expression with intact WT KCNE1 or its C-terminus (aa63-129), despite normal membrane trafficking and surface co-localization of KCNQ1 S338F and wt KCNE1. Structural modeling indicated that the S338F mutation specifically alters the interaction between the S6 domain of one KCNQ1 subunit and the S4-S5 linker of another, inhibiting voltage-induced movement synergistically with KCNE1 binding. CONCLUSIONS: A novel KCNQ1 mutation specifically impaired channel function in the presence of KCNE1. Our structural model shows that this mutation effectively immobilizes voltage gating by an inhibitory interaction that is additive with that of KCNE1. Our findings illuminate a previously unreported mechanism for LQTS, and validate recent theoretical models of the closed state of the KCNQ1:KCNE1 complex.

Cytoskeletal actin microfilaments and the transient outward potassium current in hypertrophied rat ventriculocytes.

The durations of transmembrane action potentials recorded from single myocytes isolated from the endocardial surface of hypertrophied left ventricles of rats were increased, compared to the durations recorded from normal left ventricular cells at 36-37 degrees C. Exposure to phalloidin (1-20 microM, < 20 min), a specific stabilizer of the non-myofibrillar actin microfilament component of the cardiac cytoskeleton, had no effect on action potential duration of normal cells, but significantly shortened the prolonged action potentials of hypertrophied cells. Cytochalasin D (5-50 microM), a disrupter of the actin microfilaments, also had little effect on action potential duration of normal cells. However, cytochalasin D further increased the action potential duration of hypertrophied cells at 10 min exposure. The addition of phalloidin to solutions containing cytochalasin D, reduced the latter's increase of action potential duration in hypertrophied cells. Whole-cell transient outward K(+) current (I(to1)) density was significantly decreased in hypertrophied cells. At a test potential of +60 mV, the mean I(to1) density recorded from normal cells was 13.5 +/- 1.1 pA pF(-1) (n = 18) compared to 4.17 +/- 1.2 pA pF(-1) for LVH cells (n = 22; P < 0.05). Phalloidin (20 microM) increased and cytochalasin D (50 microM) decreased whole-cell I(to1) in hypertrophied cells but had no effect on I(to1), in normal cells. When equimolar concentrations were used, phalloidin, 10 microM, reversed the decrease in I(to1) brought about by cytochalasin D, 10 microM, in hypertrophied cells. The L-type calcium current density was reduced in LVH compared to normal cells. Phalloidin (20 microM) and cytochalasin D (50 microM) had no effect on I(Ca,L) in normal or LVH myocytes. The decrease in I(to1) in hypertrophied cells and the altered I(to1) responsiveness to phalloidin and cytochalasin D reflect modification of I(to1) channel function mediated, in part, through hypertrophy-altered cytoskeletal actin microfilament regulation of I(to1).