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，启动大多数快速谷氨酸突触的神经递质释放。钙通过突触前钙通道进入，形成一个局部的高钙浓度区域，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。神经递质释放依赖于通过突触前Ca通道的三次或四次Ca电流，因此Ca进入的微小变化对突触传递有很大影响。钙依赖性突触传递的促进和抑制是神经系统信息编码和传递的重要决定因素。我们的研究结果为突触前Ca通道在突触传递和短期突触可塑性中的功能和调控提供了重要的新见解。首先，我们进一步确定了Ca通道与SNARE蛋白相互作用的分子机制，以及通过蛋白磷酸化对这种相互作用的调节。其次，我们发现钙调蛋白样神经元钙传感器（nCaS）蛋白VILIP-2通过与钙调蛋白和CaBP1在同一结合位点的相互作用调节Cav2.1通道，但具有不同的调节作用。第三，我们发现CaBP1和VILIP-2对Cav2.1通道的不同调节作用需要n端肉豆蔻酰化，并且这些nCaS蛋白的n端叶赋予了它们调节的特异性。第四，我们发现，在转染的颈上神经节（SCG）神经元突触中，ncas依赖性的Cav2.1通道的促进和失活是短期促进和抑制突触传递的主要原因，首次揭示了短期突触可塑性的分子机制。最后，我们意外地发现Ca/calmodulin依赖性蛋白激酶II （CaMKII）通过特异性结合c端结构域的一个位点来调节Cav2.1通道，这可能使激酶定位于对Ca进入和附近蛋白磷酸化的快速反应。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蛋白结合和调控Cav2.1通道的分子机制；2. 确定nCaS蛋白在短期突触可塑性中的功能；3. 探索特异性结合Cav2.1通道的CaMKII的信号功能；和4。确定突触前CaMKII在突触传递和突触可塑性中的功能作用。这些实验将为突触前Ca通道的调节以及这种调节在短期突触可塑性中的作用提供新的见解，突触可塑性是神经系统中信息编码和传递的重要形式。公共卫生相关性：神经末梢的钙通道开始突触传递过程，将信息从一个神经传递到另一个细胞，以及传递到肌肉和激素分泌细胞。这些钙通道的正常功能和调节的失败会导致癫痫、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.