Mutations in Na1.5 Reveal Calcium-Calmodulin Regulation of Sodium Channel.

Mutations in the gene, encoding the cardiac voltage-gated sodium channel Na1.5, are associated with inherited cardiac arrhythmia and conduction disease. Ca-dependent mechanisms and the involvement of β-subunit (Naβ) in Na1.5 regulation are not fully understood. A patient with severe sinus-bradycardia and cardiac conduction-disease was genetically evaluated and compound heterozygosity in the gene was found. Mutations were identified in the cytoplasmic DIII-IV linker (K1493del) and the C-terminus (A1924T) of Na1.5, 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 Na1.5 mutants were studied by pull-down and biotinylation assays. The mutation K1493del rendered Na1.5 non-conductive. Na1.5 altered the gating properties of co-expressed functional Na1.5, in a Ca and Naβ1-dependent manner. Na1.5 impaired Naβ1-dependent gating regulation. Ca-dependent CaM-interaction with Na1.5 was blunted in Na1.5. Electrical charge substitution at position 1493 did not affect CaM-interaction and channel functionality. Arrhythmia and conduction-disease -associated mutations revealed Ca-dependent gating regulation of Na1.5 channels. Our results highlight the role of Na1.5 DIII-IV linker in the CaM-binding complex and channel function, and suggest that the Ca-sensing machinery of Na1.5 involves Naβ1.