Mutations in NaV1.5 Reveal Calcium-Calmodulin Regulation of Sodium Channel

Abstract

Mutations in the SCN5A gene, encoding the cardiac voltage-gated sodium channel Na V 1.5, are associated with inherited cardiac arrhythmia and conduction disease. Ca 2+ -dependent mechanisms and the involvement of β-subunit (Na V β) in Na V 1.5 regulation are not fully understood. A patient with severe sinus-bradycardia and cardiac conduction-disease was genetically evaluated and compound heterozygosity in the SCN5A gene was found. Mutations were identified in the cytoplasmic DIII-IV linker (K1493del) and the C-terminus (A1924T) of Na V 1.5, both are putative CaM-binding domains. These mutants were functionally studied in human embryonic kidney (HEK) cells and HL-1 cells using whole-cell patch clamp technique. Calmodulin (CaM) interaction and cell-surface expression of heterologously expressed Na V 1.5 mutants were studied by pull-down and biotinylation assays. The mutation K1493del rendered Na V 1.5 non-conductive. Na V 1.5 K1493del altered the gating properties of co-expressed functional Na V 1.5, in a Ca 2+ and Na V β1-dependent manner. Na V 1.5 A1924T impaired Na V β1-dependent gating regulation. Ca 2+ -dependent CaM-interaction with Na V 1.5 was blunted in Na V 1.5 K1493del . Electrical charge substitution at position 1493 did not affect CaM-interaction and channel functionality. Arrhythmia and conduction-disease -associated mutations revealed Ca 2+ -dependent gating regulation of Na V 1.5 channels. Our results highlight the role of Na V 1.5 DIII-IV linker in the CaM-binding complex and channel function, and suggest that the Ca 2+ -sensing machinery of Na V 1.5 involves Na V β1.

Inter-Regulation of Kv4.3 and Voltage-Gated Sodium Channels Underlies Predisposition to Cardiac and Neuronal Channelopathies

“…Na v 1.5 and K v 4.3 α-subunits have always been considered to be functionally independent. However, an increasing body of evidence points to the fact that voltage-gated ion channel α-subunits may not function completely independently of each other [2,10,11,[16][17][18].The results described in the present study point to a fundamentally different model of sodium and potassium α-subunits interaction and function. We demonstrate that Na v 1.5 and K v 4.3 α-subunits interact and inter-regulate each other's function.…”

An interaction between the III-IV linker and CTD in NaV1.5 confers regulation of inactivation by CaM and FHF

“…For experiments with mutations at the interaction site (on III-IV linker and CTD) the total amount of cDNA used was 3 µg of WT or mutant Na V 1.5, 1.5 µg of β1 subunit, 2 µg of CaM or FGF12B or empty vector along with 0.5 µg of enhanced GFP (eGFP). Since a previous report suggested that the Na V β1 subunit can affect Ca 2+ /CaM-dependent regulation of Na V 1.5 (Nof et al, 2019), we tested specifically whether coexpression of Na V β1 affected Na V 1.5 persistent current under Ca 2+ chelated conditions-one of our main readouts in this study. Fig.…”

“…A K1493 deletion mutant, discovered in a large family with sudden cardiac death, was studied in HEK293 cells and found to produce decreased voltage-dependent Na + currents (Zumhagen et al, 2013). It was separately discovered in the parent of a proband with compound mutations, and in that study, mutant channels did not support Na + current (Nof et al, 2019). The critical role of E1784 and its interaction with the III-IV linker is further highlighted because E1784K is also the most commonly reported Brugada syndrome mutation (Kapplinger et al, 2015).…”

Andersen–Tawil Syndrome Is Associated With Impaired PIP2 Regulation of the Potassium Channel Kir2.1

“…Biotinylation of surface proteins was carried out essentially as described (Nof et al, 2019). Briefly, transfected HEK cells were incubated with 0.5 mg/ml EZ-Link Sulfo-NHS-SS-Biotin (Pierce, Rockford, IL, USA) in PBS pH 8, supplemented with 1 mM Ca 2+ and 0.5 mM Mg 2+ , for 30 min at 4°C.…”