Differential modulation of fast inactivation in cardiac sodium channel splice variants by Fyn tyrosine kinase.

Background/aims: Post-translational modifications such as phosphorylation and dephosphorylation can finely tune the function of ion channels. Nav1.5 is the main sodium channel in human hearts and alternative splicing of the transcript generates two major splice variants, characterized by the presence (Q-pre) or absence (Q-del) of glutamine at position 1077. In the heart, both the Nav1.5 channel and Fyn tyrosine kinase are colocalized at adherens junctions. This study aimed to investigate the modulation of the aforementioned splice variants by Fyn tyrosine kinase.

Methods And Results: Q-del and Q-pre were transiently expressed alone, with catalytically active Fyn kinase (FynKa) or with a catalytically dead Fyn kinase (FynKd). Co-expression of Nav1.5 channel splice variants and Fyn kinase was confirmed by Western blotting and their Interaction was established by co-immunoprecipitation experiments. The enzymatic activity of Fyn kinase and phosphorylation of Nav1.5 channel were ascertained by immunoprecipitation and anti-phosphotyrosine immunoblotting. Whole-cell ionic currents were recorded in patch clamp experiments to examine the modulation of Nav1.5 channel variants by Fyn kinase, which indicated a hyperpolarizing shift of 9.68 mV in fast inactivation of Q-del. In contrast, a depolarizing shift of 8.77 mV in fast inactivation was observed in the case of Q-pre, while activation curves remained unaltered for both splice variants. This differential modulation in fast inactivation was further assessed by mutating tyrosine 1495 to phenylalanine in the inactivation loop, which completely removed the modulatory effect of Fyn kinase in Q-pre splice variant, while in Q-del variant hyperpolarizing shift in fast inactivation was reduced to 4.74 mV. Finally, the modulatory effect of Fyn kinase was compensated at a mid-value of 94.63 ± 0.34, when both splice variants were co-expressed at a normal physiological ratio.

Conclusion: Q-del and Q-pre were differentially modulated by Fyn kinase, and this fine modification resulted in smooth electrical activity in the heart.