Evidence for multiple Src binding sites on the alpha1c L-type Ca2+ channel and their roles in activity regulation

Src has been proposed to activate L-type calcium channel activity by binding to the alpha1c subunit. In the II-III linker region of this subunit there is a novel consensus sequence for Src binding. We have examined whether this site is a functional Src interaction site and investigated the effect displacing Src from this region has on calcium channel activity. In vitro binding assays were performed to map alpha1 subunit interaction sites. Cardiac myocytes were isolated enzymatically from rat ventricles. Whole cell patch-clamp technique was used to record Ca(2+) channel currents in cells that had been loaded with the Src inhibitor PP1 and/or peptides with amino acid sequence corresponding to the hypothesized Src docking site. Co-immunoprecipitation and pull-down studies were undertaken to identify proteins co-complexing with the alpha1 subunit. Peptides corresponding to the II-III linker region and C-terminal tail of the alpha1c subunit, but not scrambled peptide controls, were found to inhibit Src SH3 domain binding to the channel and significantly reduced the channel current amplitude. The II-III linker region peptide shifted the inactivation curve to the left whereas the C-terminal tail region peptide shifted the activation curve to the right when compared to scramble peptide controls. PP1-pre-treatment of myocytes also reduced the current amplitude, decreased the V(1/2) for channel inactivation and abolished any further effect on currents by Src binding peptides. The tyrosine kinase PYK2 was found to co-associate with Src and the channel, but PP1 pre-treatment reduced this co-association. Src binds to both the II-III linker and C-terminal tail regions of the alpha1c subunit to differentially modulate channel activity. PYK2 is also able to co-complex with Src when bound to this region of the channel but only when Src is catalytically active. Together the two kinases may synergistically regulate channel activity.