Regulation of Vascular Tone and Ca channels by Integrins

描述（由申请人提供）：l型ca2 + (CaL)通道是血管平滑肌（VSM）中ca2 +的主要进入途径。CaL电流是肌原性张力所必需的，它提供了一个基础水平的收缩，在此基础上其他机制产生血管扩张或血管收缩。至少有三种不同的整合素调节CaL通道。整合素是细胞基质粘附受体，能够将机械力通过细胞膜传导到细胞骨架。α β 3整合素的激活抑制贴片夹紧VSM细胞的CaL电流并抑制小动脉肌张力；激活alpha5beta1可增强CaL电流并增强肌原性张力（116,18）。本研究的核心假设是β 1整合素通过非受体酪氨酸激酶c-Src和丝氨酸-苏氨酸激酶PKA对通道alc亚基的双重磷酸化的信号通路，对VSM CaL电流和小动脉张力进行急性和强直性调节。为了验证这一假设，提出了三个具体目标，重点关注alpha5beta1整合素，但包括其他几种VSM整合素：A)确定细胞内激酶在小动脉平滑肌中激活alpha5beta1整合素后CaL电流增强中的作用。B)确定其他VSM整合素（beta1和beta3）调节CaL电流和小动脉张力的机制。C)确定通过alpha5beta1整合素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，包括测量大鼠骨骼肌加压小动脉的直径，膜片钳记录天然和异种表达的VSM CaL通道，免疫印迹，磷酸化测定，定点诱变，数字成像和原子力显微镜。该结果将提供一个关于VSM CaL通道如何被整合素及其内源性细胞外基质（ECM）配体调节的更完整的图景。这些研究对正常和疾病状态下ECM对血管离子通道的调节具有广泛的意义。例如，在高血压、动脉粥样硬化和再狭窄等血管重构发生的病理状态下，血管功能会因ECM组成和整合素表达的改变而受损。此外，阐明CaL通道和β 1整合素之间的信号转导途径将为整合素和ECM如何调节离子通道建立一个通用范例。

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.