Increased tyrosine phosphorylation of alpha(1C) subunits of L-type voltage-gated calcium channels and interactions among Src/Fyn, PSD-95 and alpha(1C) in rat hippocampus after transient brain ischemia.
It has been reported that the Src family kinases-mediated tyrosine phosphorylation of alpha(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 alpha(1C) and in the interactions involving Src/Fyn, alpha(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 alpha(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 alpha(1C) subunits. More interestingly, co-immunoprecipitation analysis showed that 15 min of reperfusion dramatically increased the interaction between PSD-95 and alpha(1C) and promoted the formation of alpha(1C)-PSD-95-Src complexes, for the first time. The protein levels of alpha(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 alpha(1C) subunits by Src family kinases and may contribute to the up-regulation of L-VGCCs activity in postischemic hippocampus.
Hou
Xiao Yu
XY
Research Center for Biochemistry and Molecular Biology, Xuzhou Medical College, 84 West Huaihai Road, Xuzhou, 221002, Jiangsu, China.
Zhang
Guang Yi
GY
Yan
Jing Zhi
JZ
Liu
Yong
Y
D000818:Animals; D015220:Calcium Channels / Q000378:metabolism*; D000076122:Disks Large Homolog 4 Protein; D006624:Hippocampus / Q000378:metabolism / Q000503:physiopathology; D015151:Immunoblotting; D047908:Intracellular Signaling Peptides and Proteins; D002546:Ischemic Attack, Transient / Q000378:metabolism / Q000503:physiopathology*; D008297:Male; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D021122:Protein Subunits / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051076:Proto-Oncogene Proteins c-fyn; D051381:Rats; D015424:Reperfusion; D014443:Tyrosine / Q000378:metabolism*; D019061:src-Family Kinases / Q000378:metabolism
D015220:Calcium Channels; D000076122:Disks Large Homolog 4 Protein; C501449:Dlg4 protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D021122:Protein Subunits; D011518:Proto-Oncogene Proteins; C033652:postsynaptic density proteins; D014443:Tyrosine; C494961:Fyn protein, rat; D051076:Proto-Oncogene Proteins c-fyn; D019061:src-Family Kinases