TYROSINE KINASES AND CAPACITATIVE CALCIUM ENTRY

在大多数非兴奋性细胞中，内部Ca 2+储存的耗尽导致 质膜Ca 2+进入途径的激活， 对于补充体内钙库和维持 胞浆Ca ~（2+）浓度的长期升高。 目前该 一种机制，通过这种机制，内部储存的耗尽发出激活的信号， 这种Ca 2+进入途径还没有完全了解。 然而，我们的工作 实验室表明酪氨酸激酶在这种信号传导中起作用 并指出c-src是酪氨酸激酶的主要候选者 涉案 在这个建议中，我们描述了实验来检验假设 c-SRC是耦合Ca 2+储存耗尽的酪氨酸激酶， 激活Ca 2+进入途径。 我们会检测c-src是否参与 通过改变c-src的表达水平， 这些变化对缓激肽刺激的Ca 2+内流和Ca 2+库的影响 耗尽 我们将使用表达反义技术和成纤维细胞 从c-src阴性的转基因小鼠中检测减少c-src的作用。 src水平。 我们还将表示v-src，并确定这是否可以 在没有缓激肽刺激的情况下激活Ca ~（2+）内流 耗尽 我们将继续我们的调查刺激酪氨酸激酶 在用毒胡萝卜素耗尽Ca 2+池后的活性。 由于我们 初步的实验表明，c-src可能是响应于 Ca 2+池耗竭，我们将首先关注c-src。 我们将 免疫沉淀来自对照细胞和其Ca 2+库具有 通过各种方法被耗尽，并通过测量c-src活性， 体外激酶测定。 我们的假设是，Ca 2+库耗竭导致 激活c-src酪氨酸激酶活性。 我们描述了电生理实验来表征通道 由缓激肽激活，Ca 2+池耗竭和c-src过表达。 我们建议测试的假设，一个通道的属性类似于 Ca 2+释放激活的Ca 2+通道（Icrac），先前在 肥大细胞和淋巴细胞，在成纤维细胞中被缓激肽 刺激和Ca 2+库耗尽的机制，这取决于 c-src的激活。

In most nonexcitable cells, the depletion of internal Ca2+ stores leads to the activation of a plasma membrane Ca2+ entry pathway which plays an important role both for refilling internal Ca2+ stores and for maintaining a prolonged elevation of cytosolic Ca2+ concentration. At present, the mechanism by which depletion of internal stores signals the activation of this Ca2+ entry pathway is not fully understood. However, work from our laboratory suggests that tyrosine kinases play a role in this signalling pathway and points to c-src as a prime candidate for the tyrosine kinase involved. In this proposal, we describe experiments to test the hypothesis that c-src is the tyrosine kinase coupling Ca2+ store depletion to activation of a Ca2+ entry pathway. We will test for involvement of c-src by varying the level of c-src expression and investigating the impact of these changes on Ca2+ entry stimulated by bradykinin and Ca2+ pool depletion. We will use expression antisense techniques and fibroblasts from c-src negative, transgenic mice to test for the effect of reducing c- src levels. We will also express v-src and determine whether this can activate Ca2+ entry in the absence of bradykinin stimulation ofr Ca2+ pool depletion We will continue our investigation of the stimulation of tyrosine kinase activity following Ca2+ pool depletion with thapsigargin. Since our preliminary experiments suggest that c-src may be activated in response to Ca2+ pool depletion, we will focus initially on c-src. We will immunoprecipitate c-src from control cells and cells whose Ca2+ stores have been depleted by a variety of methods and measure c-src activity by in vitro kinase assays. Our hypothesis is that Ca2+ store depletion leads to the activation of c-src tyrosine kinase activity. We describe electrophysiological experiments to characterize the channels activated by bradykinin, Ca2+ pool depletion and overexpression of c-src. We propose to test the hypothesis that a channel with properties similar to the Ca2+-release-activated Ca2+ channel (Icrac), previously described in mast cells and lymphocytes, is activated in fibroblasts by both bradykinin stimulation and Ca2+ store depletion via a mechanism that is dependent on activation of c-src.