SCN4B-encoded sodium channel beta4 subunit in congenital long-QT syndrome

Abstract

Background: Congenital long-QT syndrome (LQTS) is potentially lethal secondary to malignant ventricular arrhythmias and is caused predominantly by mutations in genes that encode cardiac ion channels. Nearly 25% of patients remain without a genetic diagnosis, and genes that encode cardiac channel regulatory proteins represent attractive candidates. Voltage-gated sodium channels have a pore-forming alpha-subunit associated with 1 or more auxiliary beta-subunits. Four different beta-subunits have been described. All are detectable in cardiac tissue, but none have yet been linked to any heritable arrhythmia syndrome.

Methods and results: We present a case of a 21-month-old Mexican-mestizo female with intermittent 2:1 atrioventricular block and a corrected QT interval of 712 ms. Comprehensive open reading frame/splice mutational analysis of the 9 established LQTS-susceptibility genes proved negative, and complete mutational analysis of the 4 Na(vbeta)-subunits revealed a L179F (C535T) missense mutation in SCN4B that cosegregated properly throughout a 3-generation pedigree and was absent in 800 reference alleles. After this discovery, SCN4B was analyzed in 262 genotype-negative LQTS patients (96% white), but no further mutations were found. L179F was engineered by site-directed mutagenesis and heterologously expressed in HEK293 cells that contained the stably expressed SCN5A-encoded sodium channel alpha-subunit (hNa(V)1.5). Compared with the wild-type, L179F-beta4 caused an 8-fold (compared with SCN5A alone) and 3-fold (compared with SCN5A + WT-beta4) increase in late sodium current consistent with the molecular/electrophysiological phenotype previously shown for LQTS-associated mutations.

Conclusions: We provide the seminal report of SCN4B-encoded Na(vbeta)4 as a novel LQT3-susceptibility gene.

Figure 1

Clinical characterization and molecular discovery of L179F-SCN4B. A, Electrocardiographic evaluation of 21-month-old index case shows QTc=712 ms and 2:1 AV block. B, Holter evaluation of index case shows T wave alternans during 1:1 conduction. C, Pedigree; affected status was assigned as outlined previously: QTc>440 ms in males and >460 ms in females. QTc values are shown below each individual. Ages at clinical evaluation are shown above each individual. *Age at death. D, DNA sequencing chromatograms demonstrate a leucine (L) to phenylalanine (F) substitution at residue 179. Bottom, Aberrant denaturing high-performance liquid chromatography profile. Top, Wild-type.

Figure 2

Location of L179F in the β4 subunit. A, Linear topology of the β4 subunit. Numbering of the amino acid sequence is relative to the full-length gene product. B, L179F conservation across species.

Figure 3

Functional characterization of L179F in HEK293 cells stably transfected with SCN5A-encoded Nav1.5 α-subunit. A, Representative traces of sodium channel current recorded with the whole-cell configuration of the patch-clamp technique for a clamp step from −140 mV to various potentials. Current amplitude and time course are shown on each panel for the sodium channel alone, sodium channel plus WT-β4, and sodium channel plus β4 mutant. No significant differences in amplitude or current/time course were noted (Summary data in Table 2). B, Steady-state voltage dependence of activation (right plot) and inactivation (left plot) with and without WT-β4 or L179F-β4. Data are means of measurements, and the lines are Boltzmann fits with n numbers and parameters of the fit found in Table 2. C, Detail of the “window” current where the activation curves overlap the inactivation curves. L179F increases window current. I_Na indicates robust sodium current; G_Na, sodium conductance (calculated from peak sodium current [I_Na] divided by the driving force [V–Na reversal potential]).

Figure 4

Effect of coexpression of L179F-β4 mutant on late sodium current. A, Representive sodium current traces in response to a step to −60 mV for 700 ms from a holding potential of −140 mV (protocol inset) are shown. Leak-subtracted currents were normalized to peak current and are shown on a scale such that peak current is off-scale to emphasize the small late component. B, Summary data showed that L179F-β4 mutant increased late sodium current during the window of terminal repolarization as much as the α subunit sodium channel mutation (ΔKPQ) that causes LQT3.

Figure 5

Coimmunoprecipitation of SCN5A and β4 subunit. The lysates were obtained from untransfected HEK cells (column 1) and from cells cotransfected with HA-SCN5A and Myc-β4 (column 2). Columns 3 to 6 are immunoprecipitation protein complexes derived from cotransfection of HA-SCN5A and Myc-β4-WT; HA-SCN5A and Myc-β4-L179F; HA-SCN5A and untagged β4-WT; and HA-SCN5A and untagged β4-L179F, respectively. The 38-kDa line recognizes the β4 subunit, and the bands above 38 kDa show nonspecific binding. Column 1 shows no recognition in control; column 2 shows recognition of the expressed Myc label. In columns 3 and 4, the β4 subunit was recognized in the SCN5A precipitate for both WT and L179F (red arrows). In columns 5 and 6, the β4 subunit was not recognized because the complex of unlabeled SCN5A and Myc-β4 were not precipitated.