CALCIUM CHANNELS--TARGETS OF TOXICANTS AND DISEASES

钙通道——毒物和疾病的目标

• 批准号: 2734289
• 负责人: William D Atchison
• 金额: $ 26.01万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2734289
• 关键词: Xenopus oocyte; acetylcholine; autoantibody; autoimmune disorder; autoimmunity; calcium channel; dihydropyridines; electrical measurement; environmental toxicology; gene expression; genetic library; human genetic material tag; human tissue; immunoglobulin G; laboratory mouse; molecular cloning; motor neurons; nerve endings; neuromuscular disorder; neuromuscular junction; neurotransmitter transport; tissue /cell culture; voltage /patch clamp; voltage gated channel

The long-term goal of this proposal is to study the role which neuronal voltage-gated Ca2+ channels play in chemical-induced toxicity, and disease processes. These channels regulate numerous critical cellular functions including secretion of neurotransmitters and hormones, regulation of excitability, condition of action potentials in cardiac and smooth muscle as well as in dendritic regions of some neurons, growth cone elongation and gene expression; thus they are crucial to the cell signalling process. They are the targets of numerous therapeutic agents, arthropod and mullusc toxins and environmentally relevant toxic metals. These channels may also be target sites in certain diseases. Lambert-Eaton Myasthenic Syndrome (LEMS) is one such disorder. It is an autoimmune disorder characterized by decreased evoked quantial release of acetylcholine (Ach) and disruption of the presynaptic active zones, the sites at which Ach is thought to be released. LEMS antibodies are thought to be directed specifically at the Ca2+ channels located at or near the active zone. During the last funding period we showed that LEMS auntoantibodies specifically reduce the function of nerve terminal Ca2+ channels and moreover shift the pattern of CA2+ channel phenotype to one expressing marked sensitivity to dihydropyridine- (DHP) type drugs. The goals then of the present applicaiton are to identify and characterize definitively the Ca2+ channel subtypes involved in the disease process, and to determine the basis for the shift in Ca2+ channel subtype during this disorder. The specific hypothesis to be tested is that exposure of nerve terminals isolated from rat brain to serum or IgG obtained from patients iwth LEMS causes destruction of P/Q subtypes of Ca2+ channels which normally control Ach release at themotor nerve ending and subsequently cause unmasking or expression of L-type DHP-sensitive channels in the axon terminal. Molecular cloning techniques, northern blot analyses of steady-state mRNa expression from sinal motor neuros and electrophysiological recordings of nerve terminal Ca2+ currents in mice which LEMS is induced by passive transfer will be studied to examine the time course and onset of the induction of DHP sensitivity as well as determine whether increased DHP-sensitivity in LEMs occurs due to enhanced expression of new L-type channels. To examine the specific types of Ca2+ channels affected by LEMS in isolation, we propose to express cloned P- and Q-type Ca2+ channels in the Xenopus oocyte expression system. We have currently isolated segments of the alpha2sigma and beta3 subunit of Ca2+ channels from human fetal spinal cord using human fetal spinal cord RNA and isolate full length copies of these genes, sequence them and engineer them with appropriate sequences for expression studies in Xenupus oocytes for 2 microelectrode voltage clamp recordings. Results of this proposal should provide information regarding the types of Ca2+ channels affected in LEMS. It will also hopefully provide structural information regarding the types of Ca2+ channel involved in neurotransmitter release- specifically from human spinal motor neurons. Results should also provide information on how neurons respond to chronic impairment of Ca2+ channel function. This may lead to strategies for treatment of disorders of Ca2+ channel function such as those seen with certain environmental toxicants, or in LEMS.

该提议的长期目标是研究角色 神经元电压门控Ca2+通道在化学诱导的 毒性和疾病过程。 这些渠道调节了许多 关键细胞功能，包括神经递质的分泌 和激素，调节兴奋性，作用状况 心脏和平滑肌以及树突状的潜力 某些神经元的区域，生长锥伸长和基因 表达;因此它们对于细胞信号传导过程至关重要。 它们是众多治疗剂，节肢动物的目标 和mullusc毒素和与环境相关的有毒金属。 这些渠道也可能是某些疾病中的目标部位。 兰伯特 - 伊东肌无力综合征（LEMS）就是这样之一 紊乱。 这是一种自身免疫性疾病，其特征是降低 乙酰胆碱（ACH）的诱发数量释放和破坏 突触前的活动区域，即ACH的地点 被发布。 LEMS抗体被认为是定向的 特别是在活动处或附近的Ca2+通道处 区。 在最后的资金期间，我们证明了LEM 姨妈抗体专门降低了神经终端的功能 CA2+通道和更改Ca2+通道的模式 表型表达对二氢吡啶的明显敏感性的表型 （DHP）类型药物。 当前申请的目标是 确定和表征CA2+通道亚型 参与疾病过程，并确定 在这种疾病期间，Ca2+通道亚型的转移。 具体 要测试的假设是神经终末的暴露 从大鼠脑到血清或从患者获得的IWTH LEM获得的IgG 导致CA2+通道的P/Q亚型破坏 通常控制CONC在主体神经结尾处的释放和 随后引起L型的揭开或表达 轴突端子中的DHP敏感通道。 分子克隆 技术，稳态mRNA表达的北印迹分析 从辛纳尔运动神经和电生理记录 小鼠中的神经末端Ca2+电流由LEM诱导 被动转移将研究以检查时间课程和 DHP灵敏度的诱导开始并确定 LEM中DHP的敏感性是否增加。 增强了新的L型通道的表达。 检查 特定类型的Ca2+通道受到LEM的影响，我们 提议在该中表达克隆的P-和Q-Type Ca2+通道 爪蟾卵母细胞表达系统。 我们目前已经孤立了 Ca2+通道的alpha2sigma和beta3亚基的段 使用人胎儿脊髓RNA从人类胎儿脊髓 并分离这些基因的全长副本，对它们进行顺序，然后 用适当的序列为他们设计用于表达研究的序列 2个微电极电压夹记录的Xenupus卵母细胞。 该建议的结果应提供有关 LEM中影响的Ca2+通道的类型。 它也希望 提供有关CA2+类型的结构信息 涉及神经递质释放的通道 - 特别是从 人脊柱运动神经元。 结果也应提供 有关神经元如何应对慢性障碍的信息 CA2+通道功能。 这可能会导致治疗策略 Ca2+通道功能的疾病，例如看到 某些环境有毒物质或在LEM中。