Abstract
In the present study, we identified a novel splice variant of the human cardiac Na(+) channel Na(v)1.5 (Na(v)1.5d), in which a 40-amino acid sequence of the DII/DIII intracellular linker is missing due to a partial deletion of exon 17. Expression of Na(v)1.5d occurred in embryonic and adult hearts of either sex, indicating that the respective alternative splicing is neither age-dependent nor gender-specific. In contrast, Na(v)1.5d was not detected in the mouse heart, indicating that alternative splicing of Na(v)1.5 is species-dependent. In HEK293 cells, splice variant Na(v)1.5d generated voltage-dependent Na(+) currents that were markedly reduced compared with wild-type Na(v)1.5. Experiments with mexiletine and 8-bromo-cyclic AMP suggested that the trafficking of Na(v)1.5d channels was not impaired. However, single-channel recordings showed that the whole-cell current reduction was largely due to a significantly reduced open probability. Additionally, steady-state activation and inactivation were shifted to depolarized potentials by 15.9 and 5.1 mV, respectively. Systematic mutagenesis analysis of the spliced region provided evidence that a short amphiphilic region in the DII/DIII linker resembling an S4 voltage sensor of voltage-gated ion channels is an important determinant of Na(v)1.5 channel gating. Moreover, the present study identified novel short sequence motifs within this amphiphilic region that specifically affect the voltage dependence of steady-state activation and inactivation and current amplitude of human Na(v)1.5.
Highlights
In the mammalian heart, the Naϩ channel isoform Nav1.5 is thought to determine the major electrophysiological and pharmacological properties of the INa in atrial and ventricular cardiomyocytes [1, 19]
Identification of a Novel Human Nav1.5 Splice Variant—Detection of the splice variant Nav1.5a in both rat and mouse [6, 7] suggested that alternative splicing of exon 18 of the cardiac Naϩ channel is conserved among mammals
The fact that Nav1.5d appeared over a long range during ontogeny in both male and female hearts indicates that this splice variant is constantly produced in the human heart under normal physiological conditions
Summary
The reliability of the assay conditions was tested by amplification of the Nav1.5 and Nav1.5d fragments using mixtures of respective plasmids, as previously described [7]. Plasmids for expression of Nav1.5-GFP and Nav1.5d-GFP in HEK293 cells were constructed as previously described [21]. 24 h after transfection, cells were incubated in mexiletine-containing medium (500 M) for 24 h before measuring whole-cell currents. Patch clamp recordings were carried out using an internal solution containing 60.0 mM CsCl, 70.0 mM CsAsp, 11.0 mM EGTA, 1.0 mM MgCl2, 1.0 mM CaCl2, 10.0 mM HEPES, and 5.0 mM Na2ATP, pH 7.2 (CsOH). The pipette resistance was between 10 and 50 megaohms when filled with the following solution: 140.0 mM NaCl, 2.5 mM CaCl2, 4.0 mM KCl, 1.0 mM MgCl2, 5.0 mM HEPES, pH 7.4 (NaOH).
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