alpha-1-syntrophin mutation and the long-QT syndrome: a disease of sodium channel disruption

Abstract

Background: Long-QT syndrome (LQTS) is an inherited disorder associated with sudden cardiac death. The cytoskeletal protein syntrophin-alpha(1) (SNTA1) is known to interact with the cardiac sodium channel (hNa(v)1.5), and we hypothesized that SNTA1 mutations might cause phenotypic LQTS in patients with genotypically normal hNa(v)1.5 by secondarily disturbing sodium channel function.

Methods and results: Mutational analysis of SNTA1 was performed on 39 LQTS patients (QTc> or =480 ms) with previously negative genetic screening for the known LQTS-causing genes. We identified a novel A257G-SNTA1 missense mutation, which affects a highly conserved residue, in 3 unrelated LQTS probands but not in 400 ethnic-matched control alleles. Only 1 of these probands had a preexisting family history of LQTS and sudden death with an additional intronic variant in KCNQ1. Electrophysiological analysis was performed using HEK-293 cells stably expressing hNa(v)1.5 and transiently transfected with either wild-type or mutant SNTA1 and, in neonatal rat cardiomyocytes, transiently transfected with either wild-type or mutant SNTA1. In both HEK-293 cells and neonatal rat cardiomyocytes, increased peak sodium currents were noted along with a 10-mV negative shift of the onset and peak of currents of the current-voltage relationships. In addition, A257G-SNTA1 shifted the steady-state activation (V(h)) leftward by 9.4 mV, whereas the voltage-dependent inactivation kinetics and the late sodium currents were similar to wild-type SNTA1.

Conclusion: SNTA1 is a new susceptibility gene for LQTS. A257G-SNTA1 can cause gain-of-function of Na(v)1.5 similar to the LQT3.

Keywords: arrhythmia; death; ion channels; long-QT syndrome; sudden (if surviving; use heart arrest).

Figure 1

The cytoskeletal protein syntrophin α₁ (SNTA1) mutational analysis in long-QT syndrome (LQTS). Sequencing analysis demonstrates a novel nucleotide variant in SNTA1 leading to a nonsynonymous change in 3 patients (A) and amino acid conservation analysis of the A257G-SNTA1 variant (B), which modifies a highly conserved amino acid in SNTA1. C, LQT-249 family pedigree and ECG recording showing a pattern with features similar to LQT3 and a prolonged QT interval in proband III: 2, his sister III:1 and his mother II:2 bearing the A257G-SNTA1 mutation. The ECG of the proband demonstrates a prolonged QT interval and late onset T wave. D, LQT-682 family pedigree and ECG recording showing a pattern with features similar to LQT3 and a prolonged QT interval in proband II: 1 bearing the A257G-SNTA1 mutation.

Figure 2

hNa_v1.5 and cytoskeletal protein syntrophin-α₁ (SNTA1) may form a protein complex in HEK-293 cells. A, Immunohistochemical staining: The left panel shows the merged images of green fluorescence (mid) and red fluorescence (right) images. The green fluorescence depicts the GFP-tagged SNTA1 and the red fluorescence depicts the Flag-tagged hNa_v1.5. B, Coimmunoprecipitation assays. IP indicates immunoprecipitants; (+), transfected with the genes indicated in the left column; (−) nontransfected with the genes.

Figure 3

The effects of wild-type cytoskeletal protein syntrophin-α1 (WT-SNTA1) and A257G-SNTA1 on hNa_v1.5 in HEK-293 cells. A, Superimposed whole-cell current traces induced by a step-pulse protocol from a holding potential of −140 mV. B, I-V relationships. C, Voltage-dependence of peak conductance and steady-state fast inactivation. Conductance G(V) was calculated by the equation: G(V) = I/(V_m − E_rev), where I is the peak currents, E_rev is the measured reversal potential, V_m is the membrane potential. The normalized peak conductance was plotted as a function of membrane potentials. Steady-state inactivation was estimated by prepulse protocols (500 ms) from a holding potential of −140 mV. The normalized peak currents were plotted as a function of membrane potentials. Steady-state activation and inactivation were fitted with the Boltzmann equation: y = [1 + exp ((V_h − V_m)/k)]⁻¹, where y represents variables; V_h, midpoint; k, slope factor; V_m, membrane potential. The inset indicates the magnified illustration of the window currents. The solid lines represent WT-SNTA1, and the dotted lines represent A257G-SNTA1. D, Recovery from the fast-inactivation estimated by a double pulse protocol. Cells were depolarized at 0 mV for 500 ms from a holding potential of −140 mV, then stepped to −140 mV for various durations before the second pulse (20 ms at −20 mV). The fractional recovery was calculated as the ratio of peak currents at the second pulse. The recovery time course was fitted with a double exponential function: I(t)/I_max = C − A_f × exp (−t/τ_f) −A_s × exp (−t/τ_s), where t is the recovery time, A_f and A_s are the fractions of fast and slow components, τ_f and τ_s are the time constants of fast and slow components of recovery. E, Macroscopic current decay was fit with a double exponential function: I(t)/I_max = C − A_f exp(−t/τ_f) − A_s exp (−t/τ_s), where I_max is the peak current, t is the time, A_f and A_s are the fractions of fast and slow components, τ_f and τ_s are the time constants of fast and slow components, respectively. The data were represented as mean±SE.

Figure 4

The effects of wild-type cytoskeletal protein syntrophin-α₁ (WT-SNTA1) and A257G-SNTA1 on Na_v1.5 in neonatal rat cardiomyocytes. A, Superimposed whole-cell current traces induced by a step-pulse protocol from a holding potential of −140 mV. B, I-V relationships. C, Voltage-dependence of peak conductance and steady-state fast inactivation. D, Magnified current traces of the tetrodotoxin-sensitive late sodium currents induced by a long depolarization-pulse protocol (2000 ms at −20 mV from a holding potential of −140 mV). The dotted lines indicate zero current levels. E, Macroscopic current decay was fit with a double exponential function. The data were represented as mean±SE.