Alpha1-syntrophin mutations identified in sudden infant death syndrome cause an increase in late cardiac sodium current

Abstract

Background: Sudden infant death syndrome (SIDS) is a leading cause of death during the first 6 months after birth. About 5% to 10% of SIDS may stem from cardiac channelopathies such as long-QT syndrome. We recently implicated mutations in alpha1-syntrophin (SNTA1) as a novel cause of long-QT syndrome, whereby mutant SNTA1 released inhibition of associated neuronal nitric oxide synthase by the plasma membrane Ca-ATPase PMCA4b, causing increased peak and late sodium current (I(Na)) via S-nitrosylation of the cardiac sodium channel. This study determined the prevalence and functional properties of SIDS-associated SNTA1 mutations.

Methods and results: Using polymerase chain reaction, denaturing high-performance liquid chromatography, and DNA sequencing of SNTA1's open reading frame, 6 rare (absent in 800 reference alleles) missense mutations (G54R, P56S, T262P, S287R, T372M, and G460S) were identified in 8 (approximately 3%) of 292 SIDS cases. These mutations were engineered using polymerase chain reaction-based overlap extension and were coexpressed heterologously with SCN5A, neuronal nitric oxide synthase, and PMCA4b in HEK293 cells. I(Na) was recorded using the whole-cell method. A significant 1.4- to 1.5-fold increase in peak I(Na) and 2.3- to 2.7-fold increase in late I(Na) compared with controls was evident for S287R-, T372M-, and G460S-SNTA1 and was reversed by a neuronal nitric oxide synthase inhibitor. These 3 mutations also caused a significant depolarizing shift in channel inactivation, thereby increasing the overlap of the activation and inactivation curves to increase window current.

Conclusions: Abnormal biophysical phenotypes implicate mutations in SNTA1 as a novel pathogenic mechanism for the subset of channelopathic SIDS. Functional studies are essential to distinguish pathogenic perturbations in channel interacting proteins such as alpha1-syntrophin from similarly rare but innocuous ones.

Figure 1. Identification and location of SNTA1 mutations in SIDS

Depicted are the (A) DNA sequence chromatograms and (B) sequence homologies for SIDS-associated SNTA1 mutations. (C) Illustrated is the linear topology for SNTA1 with mutation localization. *Denotes previously reported LQT12-associated mutations. (D) Diagram shows SCN5A-SNTA1-nNOS-PMCA4b complex with location of interaction between SNTA1 and complex subunits.

Figure 1. Identification and location of SNTA1 mutations in SIDS

Depicted are the (A) DNA sequence chromatograms and (B) sequence homologies for SIDS-associated SNTA1 mutations. (C) Illustrated is the linear topology for SNTA1 with mutation localization. *Denotes previously reported LQT12-associated mutations. (D) Diagram shows SCN5A-SNTA1-nNOS-PMCA4b complex with location of interaction between SNTA1 and complex subunits.

Figure 2. Electrophysiological properties of cardiac sodium channel in HEK293 cells co-expressing PMCA4b, nNOS, and either WT or mutant SNTA1

(A) Representative whole-cell current traces showing increased peak I_Na associated with G460S-, T372M-, S287R-, and T262P-SNTA1. (B) Summary data of peak I_Na densities from every group. (C) Representative traces showing increased late I_Na associated with G460S-, T372M-, and S287R-SNTA1 compared with WT-SNTA1. (D) Summary data of late I_Na normalized to peak I_Na after leak subtraction. The number of tested cells is indicated above the bar. * P < 0.05 versus WT-SNTA1.

Figure 3. Voltage–dependent gating for SCN5A co-expressed with PMCA4b, nNOS, and either WT or mutant SNTA1

(A) None of the six SNTA1 mutations altered steady-state activation parameters significantly. (B) G460S-, T372M-, S287R-, and T262P-SNTA1 caused a statistically significant depolarizing shift in inactivation by 2.5-4.6 mV. The peak current activation data are replotted as a conductance (G) curve with steady-state inactivation relationships to show (C) G460S-, (D) T372M-, and (E) S287R-SNTA1 increase the overlap of these relationships. Lines represent fits to Boltzmann equations with parameters of the fit and n numbers in Table 2. The window area of each mutant (right slanted line area under curves) was significantly enhanced beyond WT (left slanted line area under curves).

Figure 4. Decay of macroscopic current and voltage dependence of inactivation fast time constants

(A) Representative normalized whole-cell current traces at -20 mV showing slower decay in G460S-, T372M, and S287R-SNTA1 compared with WT-SNTA1. (B) Compared with WT-SNTA1, G460S-, T372M-, and S287R-SNTA1 showed significantly larger fast component (τ_f) values across a wide range of test potentials from -20 mV to 10 mV except T372M, where deviations from WT were from -10 to 10 mV. * P < 0.05 versus WT-SNTA1.