Regulation of Vascular Tone and Ca channels by Integrins

描述（由申请人提供）：L型Ca 2+（CaL）通道是血管平滑肌（VSM）中的主要Ca 2+进入途径。 CaL电流是肌源性张力所必需的，提供了基础水平的收缩，在此基础上其他机制产生血管舒张或血管收缩。 至少有3种不同的整合素调节CaL通道。 整合素是细胞-基质粘附受体，能够将机械力跨细胞膜转导到细胞骨架。 α v β 3整联蛋白的激活抑制了膜片钳VSM细胞中的CaL电流并抑制了小动脉肌源性张力; α 5 β 1的激活增强了CaL电流并增强了肌源性张力（116，118）。 该建议的中心假设是β 1整合素通过涉及通道alc亚基由非受体酪氨酸激酶c-Src和丝氨酸-苏氨酸激酶PKA的双重磷酸化的信号通路急性和紧张性地调节VSM CaL电流和小动脉张力。 为了验证这一假设，提出了三个具体的目标，集中在α 5 β 1整联蛋白，但包括几个其他VSM整联蛋白：A）确定细胞内激酶在小动脉平滑肌中α 5 β 1整联蛋白激活后CaL电流增强中的作用。 B）确定其他VSM整合素（β 1和β 3）调节CaL电流和小动脉张力的机制。 C）确定通过α 5 β 1整联蛋白施加的机械力如何调节VSM CaL电流和小动脉张力。 为了实现这些目标，将使用多种方法的组合，包括大鼠骨骼肌加压小动脉的直径测量、天然和异源表达的VSM CaL通道的膜片钳记录、免疫印迹、磷酸化测定、定点诱变、数字成像和原子力显微镜。 这些结果将提供一个更完整的图片VSM钙通道是如何调节整合素和内源性细胞外基质（ECM）配体。 这些研究对ECM在正常和疾病状态下调节血管离子通道具有广泛的意义。 例如，在其中发生血管重塑的病理状况（例如高血压、动脉粥样硬化和再狭窄）中，血管功能在ECM组成和整合素表达的变化之后受损。 此外，阐明钙离子通道和β 1整合素之间的信号转导途径将建立一个一般的范例，离子通道是如何调节整合素和ECM。

DESCRIPTION (provided by applicant): 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.