Spatial association of the Cav1.2 calcium channel with α5β1-integrin

Chao J, Gui P, Zamponi GW, Davis GE, Davis MJ. Spatial association of the Cav1.2 calcium channel with alpha(5)beta(1)- integrin. Am J Physiol Cell Physiol 300: C477-C489, 2011. First published December 22, 2010; doi:10.1152/ajpcell.00171.2010.-Engagement of alpha(5)beta(1)-integrin by fibronectin (FN) acutely enhances Cav1.2 channel (Ca-L) current in rat arteriolar smooth muscle and human embryonic kidney cells (HEK293-T) expressing CaL. Using coimmunoprecipitation strategies, we show that coassociation of Ca-L with alpha(5)- or beta(1)-integrin in HEK293-T cells is specific and depends on cell adhesion to FN. In rat arteriolar smooth muscle, coassociations between CaL and alpha(5)beta(1)-integrin and between CaL and phosphorylated c-Src are also revealed and enhanced by FN treatment. Using site-directed mutagenesis of Ca-L heterologously expressed in HEK293-T cells, we identified two regions of CaL required for these interactions: 1) COOH- terminal residues Ser(1901) and Tyr(2122), known to be phosphorylated by protein kinase A (PKA) and c-Src, respectively; and 2) two proline-rich domains (PRDs) near the middle of the COOH terminus. Immunofluorescence confocal imaging revealed a moderate degree of wild-type Ca-L colocalization with beta(1)- integrin on the plasma membrane. Collectively, our results strongly suggest that 1) upon ligation by FN, CaL associates with alpha(5)beta(1)-integrin in a macromolecular complex including PKA, c-Src, and potentially other protein kinases; 2) phosphorylation of CaL at Y-2122 and/or S-1901 is required for association of CaL with alpha(5)beta(1)-integrin; and 3) c-Src, via binding to PRDs that reside in the II-III linker region and/or the COOH terminus of CaL, mediates current potentiation following alpha(5)beta(1)-integrin engagement. These findings provide new evidence for how interactions between alpha(5)beta(1)-integrin and FN can modulate CaL entry and consequently alter the physiological function of multiple types of excitable cells.