NEURONAL FUNCTIONS OF MULTIPLE TYPES OF CALCIUM CHANNELS

多种钙通道的神经元功能

• 批准号: 2265073
• 负责人: RICHARD W TSIEN
• 金额: $ 29.04万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-08-01 至 1997-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2265073
• 关键词: Anura; G protein; action potentials; alternatives to animals in research; autoradiography; bioenergetics; biological signal transduction; calcium channel; electrophysiology; hippocampus; laboratory rat; membrane proteins; molecular biology; neural conduction; neurons; neurotransmitter transport; neurotransmitters; radionuclides; site directed mutagenesis; synaptosomes; tetrodotoxin; tissue /cell culture; voltage gated channel

Voltage-dependent Ca2+ channels are vital for regulated Ca2+ entry and Ca2+ signaling in neurons. Functional studies have uncovered several types of Ca2+ channels, including L-, N-, T- and P- type, distinguished by pharmacology, electrophysiology and neuronal distribution. More recently, molecular cloning has revealed an even greater diversity arising from multiple genes and alternative splicing. The availability of cloned cDNAs for Ca2+ channel subunits opens up new approaches to molecular determinants of key Ca2+ channel features. Ca2+ channel selectivity for permanent and blocking ions. Mutagenesis experiments will probe the contributions of a hypothesized ring of glutamate residues within the pore, a putative outer ring of negatively charged residues, and other structural features. Changes in selectivity for Ca2+ and blocking cations, pore size, and ion-ion interactions will be tested. High-affinity binding of peptide toxins. omega-CTx-GVIA blocks N-type channels and omega-CTx-MVIIC is a potent blocker of BI channels highly expressed in cerebellum. What is the structural basis for the potent block? Is toxin specificity determined by residues near the outer mouth of the pore? Such questions will be addressed with chimeric constructs and point mutants. This may help further development of type-selective agents. G-proteins, gating modes and modulation of N-type channels. N-type Ca2+ channels display multiple patterns of gating (modes). Norepinephrine down-modulates Ca2+ channel entry by biasing against a mode with high open probability (p) and in favor of a low-p mode or no activity at all, modulation thought to involve G-protein. Experiments will explore what region(s) of the Ca2+ channel are required for G-protein signaling, whether the interaction is direct, and how neurotransmitter modulation, prepulse disinhibition, and spontaneous modal switching are altered by functional ablation of specific G-proteins. Interactions between Ca2+ channels and presynaptic membrane and vesicle membrane proteins. Antibodies to synaptotagmin (p65) or syntaxin (p35) are known to immunoprecipitate omega-CTx-GVIA binding sites (N-type Ca2+ channels). Do such interactions exist for other channel types? What are the molecular determinants of such interactions? What are the functional consequences of such interactions for transmitter release?

电压依赖性CA2+通道对于受管制的Ca2+进入至关重要 神经元中的Ca2+信号传导。 功能研究发现了几个 CA2+通道的类型，包括L-，N-，T-和P类型，区分 通过药理学，电生理学和神经元分布。 更多的 最近，分子克隆揭示了更大的多样性 由多个基因和替代剪接产生。 可用性 Ca2+通道亚基的克隆cDNA的开放方法 关键Ca2+通道特征的分子决定因素。 CA2+通道选择性，用于永久和阻塞离子。 诱变 实验将探测一个假设的环的贡献 孔内的谷氨酸残留物，一个假定的外环 带电残留物和其他结构特征。 选择性的变化 对于Ca2+和阻塞阳离子，孔径和离子离子相互作用将 进行测试。 肽毒素的高亲和力结合。 Omega-CTX-GVIA阻止N型 通道和omega-ctx-Mviic是BI通道的有效阻滞剂 在小脑中表达。 有效的结构基础是什么 堵塞？ 是由外口附近的残基确定的毒素特异性 毛孔？ 这些问题将通过嵌合结构解决 和点突变体。 这可能有助于进一步发展类型选择性 代理商。 G蛋白，门控模式和N型通道的调制。 N型Ca2+ 通道显示门控的多种模式（模式）。 去甲肾上腺素 通过偏向高的模式来调节Ca2+通道输入 开放概率（P），有利于低P模式或根本没有活动， 被认为涉及G蛋白的调节。 实验将探索什么 G蛋白信号传导需要CA2+通道的区域 是否直接相互作用，以及神经递质的调制如何 预硫次抑制作用和自发的模态切换被改变 特定G蛋白的功能消融。 Ca2+通道与突触前膜和囊泡之间的相互作用 膜蛋白。 突触毒素（p65）或语法素（p35）的抗体（p35） 已知可以免疫沉淀omega-ctx-GVIA结合位点（N型Ca2+ 频道）。 其他渠道类型是否存在此类交互？ 什么是 这种相互作用的分子决定因素？ 什么是功能 这种相互作用的后果释放？