Digenic inheritance novel mutations in SCN5a and SNTA1 increase late I(Na) contributing to LQT syndrome

Abstract

SCN5A and SNTA1 are reported susceptible genes for long QT syndrome (LQTS). This study was designed to elucidate a plausible pathogenic arrhythmia mechanism for the combined novel mutations R800L-SCN5A and A261V-SNTA1 on cardiac sodium channels. A Caucasian family with syncope and marginally prolonged QT interval was screened for LQTS-susceptibility genes and found to harbor the R800L mutation in SCN5A and A261V mutation in SNTA1, and those with both mutations had the strongest clinical phenotype. The mutations were engineered into the most common splice variant of human SCN5A and SNTA1 cDNA, respectively, and sodium current (INa) was characterized in human embryonic kidney 293 cells cotransfected with neuronal nitric oxide synthase (nNOS) and the cardiac isoform of the plasma membrane Ca-ATPase (PMCA4b). Peak INa densities were unchanged for wild-type (WT) and for mutant channels containing R800L-SCN5A, A261V-SNTA1, or R800L-SCN5A plus A261V-SNTA1. However, late INa for either single mutant was moderately increased two- to threefold compared with WT. The combined mutations of R800L-SCN5A plus A261V-SNTA1 significantly enhanced the INa late/peak ratio by 5.6-fold compared with WT. The time constants of current decay of combined mutant channel were markedly increased. The gain-of-function effect could be blocked by the N(G)-monomethyl-l-arginine, a nNOS inhibitor. We conclude that novel mutations in SCN5A and SNTA1 jointly exert a nNOS-dependent gain-of-function on SCN5A channels, which may consequently prolong the action potential duration and lead to LQTS phenotype.

Fig. 1.

ECG phenotype and genotype. A: pedigree and ECG tracing of the proband (arrow) and her son. The QTc of the proband exceeds 480 ms, and the QTc of the proband's son is 500 ms. B: sequence chromatograms of SCN5A and SNTA1 mutations and the amino acid conservation of R800 in SCN5A and A261 in SNTA1 across species. Left: arrow indicates the 2399G>T substitution leading to the R800L mutation in SCN5A. Right: arrow indicates the 782C>T substitution leading to the A261V mutation in SNTA1.

Fig. 2.

Electrophysiological properties of SCN5A in human embryonic kidney (HEK)293 cells coexpressing PMCA4b, neuronal nitric oxide synthase (nNOS), wild-type (WT), or mutant SCN5A and WT or mutant SNTA1. N^G-monomethyl-l-arginine (l-NMMA) was introduced into HEK293 cell culture media 6 h before testing. A: whole-cell current traces from representative experiments of each group after 24 h transfection. B: summary data of peak sodium current (I_Na) density of each group showing no significant difference of peak I_Na in R800L-SCN5A plus A261V-SNTA1, WT-SCN5A plus A261V-SNTA1, or R800L-SCN5A plus WT-SNTA1 compared with WT-SCN5A plus WT-SNTA1. Currents were elicited by test depolarization to 24 ms from a holding potential of −140 mV. Numbers of tested cells are indicated above the bar. Symbols represent means, and bars represent SE.

Fig. 3.

Late I_Na induced by cardiac sodium channel in HEK cells coexpressing PMCA4b, nNOS, WT or mutant SCN5A, and WT or mutant SNTA1. l-NMMA was introduced into HEK293 cell culture media 6 h before testing. A: representative traces showing increased late I_Na associated with R800L-SCN5A plus A261V-SNTA1 and WT-SCN5A plus A261V-SNTA1 compared with WT-SCN5A plus WT-SNTA1. B: summary data for late I_Na normalized to peak I_Na after leak subtraction. Currents were elicited by a test depolarization pulse from −140 mV to −20 mV for 700 ms after the initiation of the depolarization. Numbers of tested cells are indicated above the bar. Symbols represent means, and bars represent SE. *P < 0.05 vs. WT-SCN5A plus WT-SNTA1. See table 1 for n numbers.

Fig. 4.

Voltage-dependence of steady-state activation and inactivation was measured from HEK293 cells transiently expressing PMCA4b, nNOS, WT or mutant SCN5A, and WT or mutant SNTA1. l-NMMA was introduced into HEK293 cell culture media 6 h before testing. A: R800L-SCN5A plus A261V-SNTA1 did not alter steady-state activation parameters significantly. B: R800L-SCN5A plus A261V-SNTA1 did not affect steady-state inactivation significantly. C: peak current activation data are replotted as a conductance (G) curve with steady-stated inactivation relationships, showing that R800L-SCN5A plus A261V-SNTA1 increases the overlap of these relationships (window current) compared with WT-SCN5A plus WT-SNTA1. After incubation with l-NMMA, the increased window current of combined mutant channel was diminished. D: enlarged scale from C to better show the window current. See table 1 for n numbers.

Fig. 5.

Decay of macroscopic current. A: voltage dependence of inactivation fast time constants. When compared with WT-SCN5A plus WT-SNTA1, R800L-SCN5A plus A261V-SNTA1 showed larger fast component (τ_f) values through a wide range of test potentials from −40 mV to 10 mV. B: voltage dependence of inactivation slow time constants. When compared with WT-SCN5A plus WT-SNTA1, R800L-SCN5A plus A261V-SNTA1 showed larger slow component (τ_s) values through a wide range of test potentials from −40 mV to 10 mV. Both effects can be abolished by l-NMMA. *P < 0.05 vs. WT-SCN5A plus WT-SNTA1. See table 1 for n numbers.