Functional consequences of the arrhythmogenic G306R KvLQT1 K+ channel mutant probed by viral gene transfer in cardiomyocytes

Abstract

IKs, the slow component of the delayed rectifier potassium current, figures prominently in the repolarization of heart cells. The K+ channel gene KvLQT1 is mutated in the heritable long QT (LQT) syndrome. Heterologous coexpression of KvLQT1 and the accessory protein minK yields an IKs-like current. Nevertheless, the links between KvLQT1 and cardiac IKs are largely inferential. Since the LQT syndrome mutant KvLQT1-G306R suppresses channel activity when coexpressed with wild-type KvLQT1 in a heterologous system, overexpression of this mutant in cardiomyocytes should reduce or eliminate native IKs if KvLQT1 is indeed the major molecular component of this current. To test this idea, we created the adenovirus AdRMGI-KvLQT1-G306R, which overexpresses KvLQT1-G306R channels. In > 60 % of neonatal mouse myocytes, a sizable IKs could be measured using perforated-patch recordings (8.0 +/- 1.6 pA pF-1, n = 13). IKs was increased by forskolin and blocked by clofilium or indapamide but not by E-4031. While cells infected with a reporter virus expressing only green fluorescent protein (GFP) displayed IKs similar to that in uninfected cells, AdRMGI-KvLQT1-G306R-infected cells showed a significantly reduced IKs (2.4 +/- 1.1 pA pF-1, n = 10, P < 0.01) when measured 60-72 h after infection. Similar results were observed in adult guinea-pig myocytes (5.9 +/- 1.2 pA pF-1, n = 9, for control vs. 0.1 +/- 0.1 pA pF-1, n = 5, for AdRMGI-KvLQT1-G306R-infected cells). We conclude that KvLQT1 is the major molecular component of IKs. Our results further establish a dominant-negative mechanism for the G306R LQT syndrome mutation.

Figure 1. I_Ks in uninfected neonatal murine ventricular myocytes

A and B, representative raw current traces of slowly activating time-dependent outward currents (I_Ks) elicited by long depolarization (8 s) of neonatal mouse myocytes to +70 mV from a holding potential of -40 mV using conventional patch-clamp (A) and perforated-patch (B) techniques. C, current magnitude, measured as the difference in outward current at the beginning and end of the pulse, is plotted against time during these experiments. ▪, conventional; ○, perforated-patch recording. Rapid current run-down was observed when I_Ks was recorded using the conventional patch-clamp technique but was absent when the perforated-patch technique was employed. Cell capacitance was 42 and 25 pF, respectively, for these cells.

Figure 2. I_Ks in neonatal mouse myocytes

A, current-voltage (I-V) relationship (left panel, ▪) and raw records of a family (right panel) of I_Ks recorded from uninfected myocytes using the perforated-patch technique. B, left, the effects of 100 μm clofilium, 1 mm indapamide, 10 μm E-4031 and 10 μm forskolin + 100 μm IBMX (Forskolin) on the current amplitude of I_Ks were normalized to those measured under drug-free conditions and plotted as bar graphs (□). Right, typical I_Ks currents recorded before and after (arrows) application of these drugs. I-V relationships (left panel of A, ○) and pharmacology (left panel of B, ▪) of I_Ks recorded from AdRGI-infected cells are also shown.

Figure 3. Effects of constructs carrying the dominant-negative mutation G306R

A, bar graphs summarizing the specific inhibitory effects of transfection of pAdRMGI-KvLQT1-G306 on KvLQT1 channels. For each group of channels, current amplitudes were normalized by the mean current of the same channel type (transfected with 1 × DNA) expressed alone. Current through KvLQT1 channels increased when more pAdRMGI-KvLQT1 DNA was used for transfection but was substantially suppressed when it was co-expressed with pAdRMGI-KvLQT1-G306R. In contrast, co-expression of Kv1.3 channels with KvLQT1-G306R did not lead to current suppression compared to expression of Kv1.3 alone. Currents were measured at the end of a 4 s, +70 mV or 1 s, +60 mV pulse from a holding potential of -40 or -100 mV for KvLQT1 and Kv1.3 channels, respectively. B, distribution of raw (squares) and averaged (circles) current densities at +70 mV recorded from AdRGI- (open symbols) and AdRMGI-KvLQT1-G306R-infected (filled symbols) neonatal mouse myocytes. The latter was significantly (P < 0.05) reduced. C, top, typical current traces of I_Ks recorded during a family of stepping voltages from AdRMGI-KvLQT1-G306R-infected myocytes. Bottom, I-V relationship.

Figure 4. I_Ks in isolated adult guinea-pig ventricular myocytes

A, representative raw current traces of I_Ks recorded from control uninfected (left) and AdRMGI-KvLQT1-G306R-infected (right) myocytes. B, bar graph summarizing the current densities measured at the end of a 4 s depolarizing pulse to +60 mV. C, typical tail currents of I_Ks recorded from a control uninfected myocyte during a family of stepping voltages after a 4 s prepulse to +70 mV.