The L-type Ca 2+ (CaL) channel is the primary Ca 2+ entry pathway in vascular smooth muscle (VSM). CaL current is required for myogenic tone, providing a basal level of constriction upon which other mechanisms produce vasodilatation or vasoconstriction. At least 3 different integrins regulate the CaL channel. Integrins are cell-matrix adhesion receptors capable of transducing mechanical force across the cell membrane to the cytoskeleton. Activation of alphavbeta3 integrin inhibits CaL current in patch-clamped VSM cells and inhibits arteriolar myogenic tone; activation of alpha5beta1 potentiates CaL current and enhances myogenic tone (116,1 18). The central hypothesis of this proposal is that beta1 integrins acutely and tonically regulate VSM CaL current and arteriolar tone through a signaling pathway that involves dual phosphorylation of the channel alc subunit by the non-receptor tyrosine kinase c-Src and the serine-threonine kinase PKA. To test this hypothesis, three specific aims are proposed, focusing on alpha5beta1 integrin, but including several other VSM integrins: A) To determine the role of intracellular kinases in the potentiation of CaL current following alpha5beta1 integrin activation in arteriolar smooth muscle. B) To determine the mechanisms by which other VSM integrins (beta1 and beta3) regulate CaL current and arteriolar tone. C) To determine how mechanical forces applied through alpha5beta1 integrin regulate VSM CaL current and arteriolar tone. To accomplish these aims, a combination of approaches will be used, including diameter measurements of pressurized arterioles from rat skeletal muscle, patch clamp recordings of native and heterologously expressed VSM CaL channels, immunoblotting, phosphorylation assays, site-directed mutagenesis, digital imaging, and atomic force microscopy. The results will provide a more complete picture of how the VSM CaL channel is regulated by integrins and their endogenous extracellular matrix (ECM) ligands. These studies have wide-ranging implications for vascular ion channel regulation by ECM in both normal and disease states. For example, vascular function is compromised subsequent to changes in ECM composition and integrin expression in pathologic conditions where vessel remodeling occurs, such as hypertension, atherosclerosis and restenosis. In addition, elucidation of a signal transduction pathway between the CaL channel and beta1 integrins will establish a general paradigm for how ion channels are regulated by integrins and ECM. ? ?
