Molecular Properties of Voltage-Sensitive Calcium Channels

描述（由申请人提供）：由Cav2.1通道传导的P/Q型Ca电流负责Ca进入，该Ca进入在大多数快速突触能突触处启动神经递质释放。通过突触前Ca通道进入的Ca形成高Ca浓度的局部域，其激活附近的胞吐作用。因此，突触囊泡必须停靠在突触前Ca通道附近才能有效释放。神经递质的释放依赖于通过突触前Ca通道的Ca电流的三次方或四次方，因此Ca内流的微小变化对突触传递有很大影响。钙依赖性突触传递的易化和抑制是神经系统信息编码和传递的重要决定因素。本项目的研究结果对突触前钙通道在突触传递和短时程突触可塑性中的功能和调节提供了重要的新见解。首先，我们进一步确定了钙通道与SNARE蛋白相互作用的分子机制，以及蛋白磷酸化对这种相互作用的调节。第二，我们已经表明，钙调素样神经元钙传感器（nCaS）蛋白VILIP-2调节Cav2.1通道的相互作用在相同的结合位点的钙调素和CaBP 1，但有一个独特的一组调节效果。第三，我们已经发现，N-末端豆蔻酰化是CaBP 1和VILIP-2对Cav2.1通道的不同调节作用所必需的，并且这些nCaS蛋白的N-末端叶赋予其调节特异性。第四，我们已经发现，nCaS依赖性的Cav2.1通道的易化和失活主要负责在转染的上级颈神经节（SCG）神经元突触中的突触传递的短期易化和抑制，提供了对负责短期突触可塑性的分子机制的第一个见解。最后，我们意外地发现Ca/钙调素依赖性蛋白激酶II（CaMKII）通过特异性结合C-末端结构域上的一个位点来调节Cav2.1通道，潜在地定位激酶以快速响应Ca进入和附近蛋白的磷酸化。在下一个项目期间，我们计划在这些重要进展的基础上：1。进一步明确nCaS蛋白结合和调节Cav2.1通道的分子机制; 2.确定nCaS蛋白在短时程突触可塑性中的功能; 3.探索与Cav2.1通道特异性结合的CaMK II的信号传导功能;以及4.确定突触前CaMKII在突触传递和突触可塑性中的功能作用。这些实验将提供新的见解突触前钙通道的调节和这种调节在短期突触可塑性，在神经系统中的信息编码和传输的一种重要形式的作用。公共卫生相关性：神经末梢中的钙通道开始突触传递的过程，该过程将信息从一个神经传递到另一个神经，以及传递到肌肉和神经分泌细胞。这些钙通道的正确功能和调节的失败有助于癫痫、偏头痛、共济失调和其他神经系统疾病。我们提出的研究将为这些突触前钙通道的调节及其在短期突触可塑性中的功能提供新的见解，突触可塑性是神经系统中正常编码和信息传递的重要过程，也是神经系统疾病的靶点。

DESCRIPTION (provided by applicant): P/Q-type Ca currents conducted by Cav2.1 channels are responsible for the Ca entry that initiates neurotransmitter release at most fast glutamatergic synapses. Ca entering through presynaptic Ca channels forms a local domain of high Ca concentration that activates exocytosis in the near vicinity. Therefore, synaptic vesicles must dock near presynaptic Ca channels to be efficiently released. Neurotransmitter release is dependent on the third or fourth power of the Ca current through the presynaptic Ca channels, so small changes in Ca entry have large effects on synaptic transmission. Ca-dependent facilitation and depression of synaptic transmission is an important determinant of information coding and transmission in the nervous system. Our results in the present project period have given important new insights into the function and regulation of presynaptic Ca channels in synaptic transmission and short-term synaptic plasticity. First, we have further defined the molecular mechanism for interaction of Ca channels with SNARE proteins and the regulation of that interaction by protein phosphorylation. Second, we have shown that the calmodulin-like neuronal Ca sensor (nCaS) protein VILIP-2 regulates Cav2.1 channels by interaction at the same binding site as calmodulin and CaBP1, but has a distinct set of regulatory effects. Third, we have found that N-terminal myristoylation is required for the distinct regulatory effects of CaBP1 and VILIP-2 on Cav2.1 channels and that the N-terminal lobe of these nCaS proteins confers their specificity of regulation. Fourth, we have discovered that nCaS-dependent facilitation and inactivation of Cav2.1 channels is primarily responsible for short-term facilitation and depression of synaptic transmission in transfected superior cervical ganglion (SCG) neuron synapses, providing the first insight into the molecular mechanisms responsible for short-term synaptic plasticity. Finally, we have found unexpectedly that Ca/calmodulin-dependent protein kinase II (CaMKII) regulates Cav2.1 channels by specific binding to a site on the C-terminal domain, potentially positioning the kinase for rapid response to Ca entry and phosphorylation of nearby proteins. In the next project period, we plan to build on these important advances to: 1. further define the molecular mechanisms of binding and regulation of Cav2.1 channels by nCaS proteins; 2. determine the functions of nCaS proteins in short-term synaptic plasticity; 3. explore the signaling functions of CaMKII specifically bound to Cav2.1 channels; and 4. determine the functional role of presynaptic CaMKII in synaptic transmission and synaptic plasticity. These experiments will provide novel insights into the regulation of presynaptic Ca channels and the role of this regulation in short-term synaptic plasticity, an essential form of information encoding and transmission in the nervous system. PUBLIC HEALTH RELEVANCE: Calcium channels in nerve terminals begin the process of synaptic transmission, which communicates information from one nerve to cell to another as well as to muscle and hormone-secreting cells. Failure of correct function and regulation of these calcium channels contributes to epilepsy, migraine, ataxia, and other neurological diseases. Our proposed research will provide novel insights into the regulation of these presynaptic calcium channels and their function in short-term synaptic plasticity, an essential process for normal coding and transmission of information in the nervous system and a target for neurological disease.