A sodium channel pore mutation causing Brugada syndrome

Abstract

Background: Brugada and long QT type 3 syndromes are linked to sodium channel mutations and clinically cause arrhythmias that lead to sudden death. We have identified a novel threonine-to-isoleucine missense mutation at position 353 (T353I) adjacent to the pore-lining region of domain I of the cardiac sodium channel (SCN5A) in a family with Brugada syndrome. Both male and female carriers are symptomatic at young ages, have typical Brugada-type electrocardiogram changes, and have relatively normal corrected QT intervals.

Objectives: To characterize the properties of the newly identified cardiac sodium channel (SCN5A) mutation at the cellular level.

Results: Using whole-cell voltage clamp, we found that heterologous expression of SCN5A containing the T353I mutation resulted in 74% +/- 6% less peak macroscopic sodium current when compared with wild-type channels. A construct of the T353I mutant channel fused with green fluorescent protein failed to traffic properly to the sarcolemma, with a large proportion of channels sequestered intracellularly. Overnight exposure to 0.1 mM mexiletine, a Na(+) channel blocking agent, increased T353I channel trafficking to the membrane to near normal levels, but the mutant channels showed a significant late current that was 1.6% +/- 0.2% of peak sodium current at 200 ms, a finding seen with long QT mutations.

Conclusions: The clinical presentation of patients carrying the T353I mutation is that of Brugada syndrome and could be explained by a cardiac Na(+) channel trafficking defect. However, when the defect was ameliorated, the mutated channels had biophysical properties consistent with long QT syndrome. The lack of phenotypic changes associated with the long QT syndrome could be explained by a T353I-induced trafficking defect reducing the number of mutant channels with persistent currents present at the sarcolemma.

Figure 1

(A) Family pedigree and representative ECG tracings. All affected individuals (darkened squares for males or circles for females) carry the T353I mutation and manifest ECG changes including ST segment elevation in standard precordial leads V1-V3. The arrow identifies the proband. (B) Putative structure of the voltage-gated sodium channel showing the position of the T353I in the first pore lining segment.

Figure 2

Representative phase contrast and fluorescent images of HEK cells transfected with the SCN5a and GFP on a bicistronic vector. Control HEK cells showed no fluorescence. HEK cells transfected with a vector containing the coding sequences of either wild-type (WT) or mutant (T353I) Na⁺ channels and green fluorescent protein (GFP) showed a high percentage of fluorescent cells. Cells transfected with the mutant T353I channel tended to be larger than native cells. For these pictures, cells were plated at similar density 48 hr prior to imaging.

Figure 3

A summary of the effects of the T353I mutation. Panel A summarizes the results of patch-clamp current recordings of wild-type (WT) and T353I channels expressed in HEK cells. Cells expressing T353I demonstrated little (lower traces) or no current (upper traces). Cells expressing T353I and incubated overnight with mexiletine express significantly more current (T353I + Mexiletine). Panel B compares peak whole cell Na⁺ current normalized to cell capacitance. Peak current was significantly reduced for T353I (50 ± 12 pA/pF) compared to WT (212 ± 23 pA/pF; p<0.01) but was largely restored when T353I expressing cells were treated with mexiletine. Panel C compares the late current of cells expressing WT and T353I channels after exposure to mexiletine (T353I + Mex). Cells expressing T353I and treated with mexiletine showed a persistent late current at 200 ms (1.6 ± 0.2%) compared to WT (0.03 ± 0.02%; p<0.01). Panel D compares current-voltage relationships for WT and T353I with or without preceding mexiletine treatment showing a decreased peak current for T353I without changes in the reversal potential. Mexiletine resulted in nearly full recover of current. Panel E compares steady state activation and inactivation curves. Steady state activation parameters for WT, T353I and T353I+mex were V_½ =-50 ± 0.4, k=4.6 ± 0.4 (n=15) and V_½ =-49 ± 0.8, k=4.7 ± 0.3 (n=8), V_½ =-54 ± 1.3, k= 1.5 ± 0.5 (n=7), respectively. Steady state inactivation parameters for WT, T353I, T353I+mex were V_½ =-98 mV ± 2.2 mV, k=8.7 ± 1.9 (n=15) and V_½ =-87 ± 0.3 mV, k=8.1 ± 0.2 (n=8), V_½ =-84 ± 0.3 mV, k=7.1 ± 0.2 (n=6), respectively. Panel F demonstrates that T353I results in a trafficking defect that can be ameliorated by mexiletine. The WT SCN5a-GFP fusion construct was evenly distributed on the membrane surface. In contrast, the T353I-GFP channels appear sequestered within the cell. Overnight incubation with mexiletine resulted in mutant channel localization to the cell membrane. In each micrograph, the calibration bar represents 10 micrometers.

Figure 4

Computer modeling of action potentials (APs) of cells expressing only wild-type Na⁺ channels (solid line), a 1:1 (i.e., heterozygous) mixture of mutant (MT) and wild-type (WT) channels (dashed line), or a mixture of MT and WT channels after simulated rescue with mexiletine (bold line). The decreased inward current from MT channels reduced the AP upstroke velocity from 371 V/s to 232 V/s and the sustained inward current slightly prolonged AP duration. Upon simulated rescue of all MT channels (normal I_Na amplitude) and without continued drug present, the AP was significantly prolonged and an early afterdepolarization was generated.