Partial expression defect for the SCN5A missense mutation G1406R depends on splice variant background Q1077 and rescue by mexiletine

Department of Medicine, University of Wisconsin–Madison, Madison, Wisconsin, United States
AJP Heart and Circulatory Physiology (Impact Factor: 3.84). 11/2006; 291(4):H1822-8. DOI: 10.1152/ajpheart.00101.2006
Source: PubMed


Mutations in the cardiac Na(+) channel gene SCN5A cause loss of function and underlie arrhythmia syndromes. SCN5A in humans has two splice variants, one lacking a glutamine at position 1077 (Q1077del) and one containing Q1077. We investigated the effect of splice variant background on loss of function and rescue for G1406R, a mutation reported to cause Brugada syndrome. Mutant and wild-type (WT) channels in both backgrounds were transfected into HEK-293 cells and incubated for up to 72 h with and without mexiletine. At 8 h, neither current nor cell surface expression was observed for the mutant in either background, but both were present in WT channels. At 24 h, small (<10% compared with WT) currents were noted and accompanied by cell surface expression. At 48 h, current density was approximately 40% of WT channels for the mutant in the Q1077del variant background but remained at <10% of WT channels in Q1077. Current levels were stable by 72 h. Coexpression with beta(1)- or beta(3)-subunits or insertion of the polymorphism H558R in the background did not significantly affect current expression. Mexiletine restored current density of the mutant channel in both backgrounds to nearly WT levels. The mutant channels also showed a negative shift in inactivation, slower recovery, and enhanced slow inactivation, consistent with a loss of function phenotype. These data show that a trafficking defect may be partial and time dependent and may differ with the splice variant background. Also, expression defects and gating abnormalities may contribute to loss of function for the same mutation.

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    • "splice variants may be of significant functional relevance for disease expressivity in sodium channelopathy , as evidenced by the differential effect of these variants on reduced sodium channel membrane expression due to defective trafficking of the Brugada syndrome mutation SCN5A - G1406R ( Tan et al . 2006 ) . Moreover , SCN5A splicing is also developmentally regulated , and a specific neonatal isoform has been identified which is down - regulated after birth ( Chioni et al . 2005 ; Schroeter et al . 2010 ) . Sodium channel dysfunction may be greatly enhanced in the setting of this neonatal SCN5A splice iso - form , as demonstrated recent"
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    ABSTRACT: Over the last two decades, an increasing number of SCN5A mutations have been described in patients with long QT syndrome type 3 (LQT3), Brugada syndrome, (progressive) conduction disease, sick sinus syndrome, atrial standstill, atrial fibrillation, dilated cardiomyopathy, and sudden infant death syndrome (SIDS). Combined genetic, electrophysiological and molecular studies have provided insight into the dysfunction and dysregulation of the cardiac sodium channel in the setting of SCN5A mutations identified in patients with these inherited arrhythmia syndromes. However, risk stratification and patient management is hindered by the reduced penetrance and variable disease expressivity in sodium channelopathies. Furthermore, various SCN5A-related arrhythmia syndromes are known to display mixed phenotypes, known as cardiac sodium channel overlap syndromes. Determinants of variable disease expressivity, including genetic background and environmental factors, are suspected but still largely unknown. Moreover, it has become increasingly clear that sodium channel function and regulation is more complicated than previously assumed, and the sodium channel may play additional, as of yet unrecognized roles in cardiac structure and function. Development of cardiac structural abnormalities secondary to SCN5A mutations has been reported, but the clinical relevance and underlying mechanisms are unclear. Increased insight into these issues would enable a major next step in research related to cardiac sodium channel disease, ultimately enabling improved diagnosis, risk stratification and treatment strategies.
    The Journal of Physiology 07/2013; 591(17). DOI:10.1113/jphysiol.2013.256461 · 5.04 Impact Factor
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    • "pcDNA3.1 included a FLAG tag (sequence: DYKDDDDK between prolines P154 and P155 of SCN5A) which has been previously shown not to alter the Nav1.5 current properties [14], [15]). "
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    ABSTRACT: Brugada syndrome (BrS) is a life-threatening, inherited arrhythmogenic syndrome associated with autosomal dominant mutations in SCN5A, the gene encoding the cardiac Na(+) channel alpha subunit (Na(v)1.5). The aim of this work was to characterize the functional alterations caused by a novel SCN5A mutation, I890T, and thus establish whether this mutation is associated with BrS. The mutation was identified by direct sequencing of SCN5A from the proband's DNA. Wild-type (WT) or I890T Na(v)1.5 channels were heterologously expressed in human embryonic kidney cells. Sodium currents were studied using standard whole cell patch-clamp protocols and immunodetection experiments were performed using an antibody against human Na(v)1.5 channel. A marked decrease in current density was observed in cells expressing the I890T channel (from -52.0±6.5 pA/pF, n = 15 to -35.9±3.4 pA/pF, n = 22, at -20 mV, WT and I890T, respectively). Moreover, a positive shift of the activation curve was identified (V(1/2) = -32.0±0.3 mV, n = 18, and -27.3±0.3 mV, n = 22, WT and I890T, respectively). No changes between WT and I890T currents were observed in steady-state inactivation, time course of inactivation, slow inactivation or recovery from inactivation parameters. Cell surface protein biotinylation analyses confirmed that Na(v)1.5 channel membrane expression levels were similar in WT and I890T cells. In summary, our data reveal that the I890T mutation, located within the pore of Na(v)1.5, causes an evident loss-of-function of the channel. Thus, the BrS phenotype observed in the proband is most likely due to this mutation.
    PLoS ONE 01/2013; 8(1):e53220. DOI:10.1371/journal.pone.0053220 · 3.23 Impact Factor
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    • "These mutations can be found in all parts of the channel protein [115]. Reported mutations in the P-loops that result in gating alterations giving rise to Brugada syndrome are: G1406R (domain III) [74], D1714G (domain IV) [90], S1710L (domain IV) [86,116]. Interestingly, the only gating alteration which is common to all reports is an enhancement of slow inactivation, which underscores the importance of the outer vestibule in the modulation of this kinetic state. "
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    ABSTRACT: The outer vestibule of voltage-gated Na(+) channels is formed by extracellular loops connecting the S5 and S6 segments of all four domains ("P-loops"), which fold back into the membrane. Classically, this structure has been implicated in the control of ion permeation and in toxin blockage. However, conformational changes of the outer vestibule may also result in alterations in gating, as suggested by several P-loop mutations that gave rise to gating changes. Moreover, partial pore block by mutated toxins may reverse gating changes induced by mutations. Therefore, toxins that bind to the outer vestibule can be used to modulate channel gating.
    Marine Drugs 04/2010; 8(4):1373-93. DOI:10.3390/md8041373 · 2.85 Impact Factor
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