Differential Modulation of Fast Inactivation in Cardiac Sodium Channel Splice Variants by Fyn Tyrosine Kinase

Abstract

Background/Aims: Post-translational modifications such as phosphorylation and dephosphorylation can finely tune the function of ion channels. Na_v1.5 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 Na_v1.5 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 Na_v1.5 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 Na_v1.5 channel were ascertained by immunoprecipitation and anti-phosphotyrosine immunoblotting. Whole-cell ionic currents were recorded in patch clamp experiments to examine the modulation of Na_v1.5 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.

Identification of phosphorylation sites and binding pockets for modulation of NaV1.5 channel by Fyn tyrosine kinase

“…Phosphorylated tyrosine at position 1889 (pYEPI) can also serve as a binding site for Fyn kinase SH 2 domain, since it closely resembles to the consensus site (pYEEI) recognized by SH 2 domain [15,30]. This multisite phosphorylation of tyrosine residues in Na V 1.5 was anticipated previously [16,17] and evidenced in Na V 1.2 channel [15]. There are three binding sites in Na V 1.2; one proline-rich site for SH 3 (P 636 ILP 639 ) and two phosphorylated tyrosines for SH 2 (66 and 1893) domain.…”

Altered membrane rigidity via enhanced endogenous cholesterol synthesis drives cancer cell resistance to destruxins

“…Ionophoric activity of dtxA, dtxB and dtxE was determined by patch clamp experiments, performed as previously described [ 20 , 50 ]. Briefly, ionic currents were recorded in whole-cell mode at RT.…”

Phosphorylation of cardiac voltage‐gated sodium channel: Potential players with multiple dimensions

“…In cardiomyocytes Fyn kinase regulates stability of adherens junctions and is also localized at caveolae along with Na V 1.5 channels . Interaction of Fyn kinase with Na V 1.5 has been established where it co‐immunoprecipitates and phosphorylates the Na V 1.5 channel . This phosphorylation creates a depolarizing shift in steady‐state fast inactivation, increases recovery from inactivation and decreases rate of entry into slow inactivation .…”

“…Interaction of Fyn kinase with Na V 1.5 is complex and involves multiple steps. Binding of Fyn kinase to proline‐rich regions in the ICL I‐II domain and C‐terminus is followed by phosphorylation of nearby tyrosine residues in the N‐terminus (Y 68 , Y 87 , and Y 112 ), ICL III‐IV (Y 1494 , Y 1495 ) domain and C‐terminus (Y 1811 , Y 1889 ), indicating a complex and multistep modulation …”