Novel C-terminus frameshift mutation, 1122fs/147, of HERG in LQT2: additional amino acids generated by frameshift cause accelerated inactivation.

OBJECTIVE: The function of the C-terminus region of the human ether-a-go-go related gene (HERG) has not been well characterized except for its involvement in trafficking. To understand further the role of C-terminus region, we performed a functional analysis of a novel frameshift mutation (1122fs/147) identified in a Japanese long QT syndrome 2 (LQT2) patient who had recurrent episodes of syncope. METHODS: Wild type (WT) and mutant HERG plasmids were transfected into human embryonic kidney (HEK-293) cells, and whole-cell current was recorded by the patch-clamp technique. Confocal microscopy was performed to examine the membrane distribution of channel protein using a green fluorescent protein tagged to the N-terminus of HERG. RESULTS: The mutant 1122fs/147 alone could express current, but reduced density by 74% of control. No dominant negative effect was noted with co-expression of WT and 1122fs/147. Activation and deactivation time constants were not changed, while inactivation was accelerated in 1122fs/147 compared to WT, and V(1/2) of steady-state inactivation curve shifted by 11 mV in the negative direction. Current density of 1123stop mutant revealed 49% reduction compared to WT and showed no shift in steady-state inactivation. Confocal microscopy revealed reduced protein expression on the cell surface both in 1122fs/147 and 1123stop mutants compared to WT. CONCLUSION: Frameshift mutation at the C-terminus region with additional 147 amino acids evoked a loss of function of the HERG channel. A negative shift in steady-state inactivation induced by the additional 147 amino acids and trafficking defect contribute to a reduced current amplitude of 1122fs/147.

Effects of Na+ channel blocker, pilsicainide, on HERG current expressed in HEK-293 cells.

PURPOSE: Pilsicainide, classified as a relatively pure Na+ channel blocker, occasionally causes QT prolongation, suggesting inhibitory actions on K+ currents. We studied effects of pilsicainide on the K+ channel current of the human ether-a-go-go-related gene (HERG) in heterologous expression system. METHODS: The Patch-clamp technique in whole-cell configuration was used to record the channel current of HERG stably expressed in HEK293 cells. RESULTS: Pilsicainide suppressed peak currents of HERG channel during depolarizing pulses and tail currents upon repolarization. Pilsicainide blocked HERG current with IC50 = 20.4 microM and Hill coefficient = 0.98. Voltage-dependent activation was shifted in a negative direction by approximately 10 mV by 10 to 20 microM pilsicainide. Block increased with depolarization to voltages between -20 and 0 mV and reached the maximum level at positive voltages to 0 mV without further increase. Following drug equilibration for 10 minutes (holding potential at -100 mV), the peak outward current upon the first depolarization showed time-dependent block; tail current block was maximal. Frequency-dependent block evaluated from tail current was absent with pulse frequencies of 1.33, 0.5, and 0.2 Hz. After a steady state block was achieved, time course of current activation and deactivation was slowed by pilsicainide, and steady-state inactivation and time course of fast inactivation were mildly affected. CONCLUSIONS: Pilsicainide blocks HERG current with a preferential affinity, at least, to the open state of the channels with a fast access to binding sites.

Block of recombinant KCNQ1/KCNE1 K+ channels (IKs) by intracellular Na+ and its implications on action potential repolarization.

I(Ks), the slow component of delayed rectifier K+ current, plays an important role for the repolarization of ventricular action potential. We investigated the block of I(Ks) by intracellular Na+ ([Na+](i)), using a heterologous expression system (KCNQ1/KCNE1 expressed in COS7 cells), since this well-known blocking action on various K+ channels has not been fully or quantitatively characterized in I(Ks) current. The Na+ block of I(Ks) was concentration- and voltage-dependent and was described by a conventional binding-site model (Woodhull AM: J Gen Physiol 61: 687-708, 1973). In physiological ionic conditions, the blocking action was operating noticeably with Delta ("electrical" distance of the block site) approximately 0.6 and K(d)(0) (apparent dissociation constant at 0 mV) approximately 300 mM. Because K(d)(0) was a function of intra- and extracellular K+ concentrations, changes in ionic environments not only of [Na+](i), but also of [K+](o), affected the amplitude of I(Ks) through the modulation of the Na+ block. Based on these experimental data, we analyzed the effects of Na+ block on action potentials by a computer simulation study, using the Luo-Rudy model. In a physiological ionic environment, the Na+ block of I(Ks) contributed little to modifying action potentials. However, when action potential duration (APD) was marginally prolonged because of decreased I(Ks), as observed in M cells under the conditions of bradycardia and low [K+](o), the Na+ block of I(Ks) may contribute to arrhythmogenesis through the facilitation of early afterdepolarizations (EADs).