Increased tyrosine phosphorylation of α 1C subunits of L-type voltage-gated calcium channels and interactions among Src/Fyn, PSD-95 and α 1C in rat hippocampus after transient brain ischemia

Abstract

It has been reported that the Src family kinases-mediated tyrosine phosphorylation of α 1C 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 α 1C and in the interactions involving Src/Fyn, α 1C 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 α 1C 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 α 1C subunits. More interestingly, co-immunoprecipitation analysis showed that 15 min of reperfusion dramatically increased the interaction between PSD-95 and α 1C and promoted the formation of α 1C-PSD-95-Src complexes, for the first time. The protein levels of α 1C, 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 α 1C subunits by Src family kinases and may contribute to the up-regulation of L-VGCCs activity in postischemic hippocampus.