Increased tyrosine phosphorylation of α1C subunits of L-type voltage-gated calcium channels and interactions among Src/Fyn, PSD-95 and α1C in rat hippocampus after transient brain ischemia

It has been reported that the Src family kinases-mediated tyrosine phosphorylation of α1C subunits of L-type voltage-gated calcium channels (L-VGCCs) potentiates the channel currents. In this study, we evaluated the alterations in the tyrosine phosphorylation level of α1C and in the interactions involving Src/Fyn, α1C and PSD-95 in the hippocampus after transient (15 min) brain ischemia followed by various times of reperfusion using immunoprecipitation and immunoblotting. Transient brain ischemia was induced by the method of four-vessel occlusion in Sprague–Dawley rats. The tyrosine phosphorylation level of α1C subunits elevated immediately after brain ischemia. The elevation in phosphorylation sustained for at least 6 h and peaked at 15 min of reperfusion. Transient brain ischemia and reperfusion also caused rapid and sustained increases in the interactions of Src and Fyn with α1C subunits. More interestingly, co-immunoprecipitation analysis showed that 15 min of reperfusion dramatically increased the interaction between PSD-95 and α1C and promoted the formation of α1C-PSD-95-Src complexes, for the first time. The protein levels of α1C, Src, Fyn and PSD-95 showed no differences at all time points. These results suggest a novel mechanism involving the ischemia/reperfusion-induced recruitment of L-VGCCs, Src and Fyn to the PSD-95 signaling complex that facilitates the tyrosine phosphorylation of α1C subunits by Src family kinases and may contribute to the up-regulation of L-VGCCs activity in postischemic hippocampus.