Localization of voltage-gated Ca2+ channels and Ca2+-gated K+ channels to specific 'Active Zone Material' macromolecules at presynaptic active zones and how that influences neurotransmitter secretion

电压门控 Ca2 通道和 Ca2 门控 K 通道对突触前活性区特定“活性区物质”大分子的定位及其如何影响神经递质分泌

• 批准号: 9789983
• 负责人: Joseph Szule
• 金额: $ 7.43万
• 依托单位: TEXAS A&M UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-30 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9789983
• 关键词: 3-Dimensional; Adult; Affect; Aging; Applications Grants; Binding Proteins; Cell membrane; Charybdotoxin; Chemical Models; Chemical Synapse; Chemicals; Conotoxin; Coupling; Cytosol; Data; Development; Disease; Distal; Docking; Event; Exhibits; Exposure to; Fluorescence Microscopy; Freeze Fracturing; Goals; Gold Colloid; Homologous Protein; Human; In Situ; Knowledge; Label; Lambert-Eaton Myasthenic Syndrome; Lead; Link; Membrane; Membrane Fusion; Membrane Potentials; Methods; Modeling; Molecular; Molecular Structure; Morphology; Nervous system structure; Neuromuscular Junction; Neuromuscular Junction Diseases; Neurons; Neurotransmitters; Oral cavity; Organelles; Physiologic pulse; Physiology; Play; Positioning Attribute; Potassium Channel; Presynaptic Terminals; Probability; Proteins; Rana; Research; Resolution; Rest; Role; Site; Streptavidin; Structure; Synapses; Synaptic Transmission; Synaptic Vesicles; Time; Tissue Stains; Toxin; Trauma; United States National Institutes of Health; Vesicle; Work; analytical method; electron tomography; experimental study; fluorescence imaging; iberiotoxin; innovation; insight; macromolecule; member; molecular scale; novel; presynaptic; response; stem; synaptotagmin; transmission process; voltage

Synaptic impulse transmission fundamentally relies on the coupling of neuron impulses with neurotransmitter secretion from specialized sites along the presynaptic plasma membrane (PM) of the axon terminals called ac- tive zones. Active zones of all synapses have comparable organelles, called ‘Active Zone Material’ (AZM), which are composed of homologous proteins that assemble to form distinct classes of AZM macromolecules; AZM regulates the events that lead to neurotransmitter secretion from docked synaptic vesicles (SV) (i.e. SVs held in contact with the PM). Determining the identity of the proteins that assemble to form the AZM is neces- sary to understand the general rules that govern the molecular mechanisms that regulate neurotransmitter se- cretion throughout the nervous system under normal, experimental and disease conditions. The arrival of an electrical impulse at an active zone causes voltage-gated Ca2+ (CaV) channels to open and allow Ca2+ to enter the cytosol which results in elevated concentrations of Ca2+ near the mouth of the channel for a very brief peri- od of time. If sufficient concentrations of Ca2+ interact with the SV protein synaptotagmin it triggers membrane fusion and neurotransmitter secretion, which is the defining stage for the described impulse-secretion coupling. The Ca2+ that enters the cytosol also activates Ca2+-gated K+ (KCa) channels to repolarize the PM and deacti- vate the CaV channels to arrest further neurotransmitter secretion. Thus, the relative proximity of CaV channels to docked SVs and KCa channels strongly influences impulse-secretion coupling. In axon terminals of a model synapse, frog neuromuscular junction, it has long been suspected that both CaV and KCa channels are compo- nents of the macromolecules that span the PM at active zones arranged in parallel double row arrays de- scribed in freeze-fracture replicas. Previous studies from our lab used electron tomography to quantitatively study the 3D macromolecular structure of AZM at frog neuromuscular junctions and found that the members of a particular class of AZM macromolecules called pegs are connected to the macromolecules that span the PM. We also found that docked SVs that had the greatest probability of fusing with the PM when an impulse arrives were associated with pegs in the row proximal to the SVs that were displaced closer to them. We proposed that the proximal pegs were connected to CaV channels because the closer the CaV channel is to synaptotag- min when the impulse causes the channel to open and allow an influx of Ca2+ into the cytosol, the higher the concentration of Ca2+ exposure to synaptotagmin and the greater the probability that it will trigger membrane fusion. The objective of the research proposed here is to localize the CaV and KCa channels at active zones of frog neuromuscular junctions with sufficient resolution to determine if they are associated with the pegs that are connected to the macromolecules that span the PM, and if they are, to determine which row each channel is concentrated. To meet this objective, an innovative method involving histochemical labeling of CaV and KCa channels together with quantitative electron tomography will be used.

突触冲动的传递从根本上依赖于神经元冲动与神经递质的耦合 从轴突末梢的突触前质膜（PM）沿着的专门位点分泌，称为ac- 动态区域。所有突触的活动区都有类似的细胞器，称为“活动区物质”（AZM）， 其由组装形成不同类别的AZM大分子的同源蛋白质组成; AZM调节导致神经递质从对接的突触囊泡（SV）（即SV）分泌的事件 与PM保持联系）。确定组装形成AZM的蛋白质的身份是必要的。 了解控制调节神经递质的分子机制的一般规则， 在正常、实验和疾病条件下，整个神经系统的分泌。的到来 在活动区的电脉冲导致电压门控Ca 2+（CaV）通道打开并允许Ca 2+进入 细胞质导致通道口附近Ca 2+浓度升高，持续非常短暂的时间， 时间。如果足够浓度的Ca 2+与SV蛋白突触结合蛋白相互作用， 融合和神经递质分泌，这是所述冲动-分泌偶联的定义阶段。 进入胞质的Ca ~（2+）也激活Ca ~（2+）门控的K ~+（KCa）通道，使PM恢复，使PM失活。 激活CaV通道以阻止进一步的神经递质分泌。因此，CaV通道的相对接近性 停靠SV和KCa通道强烈影响冲动分泌耦合。在模型的轴突终末 突触，青蛙神经肌肉接头，长期以来一直怀疑CaV和KCa通道都是复合的， 在以平行双行阵列布置的活性区处跨越PM的大分子的量 在冷冻断裂复制品上刻下我们实验室以前的研究使用电子断层扫描定量地 研究了蛙神经肌肉接头处AZM的三维大分子结构，发现AZM的成员 被称为钉的一类特殊AZM大分子与跨越PM的大分子相连。 我们还发现，当脉冲到达时，对接的SV与PM融合的可能性最大 与SV近端行中的立柱相关，这些立柱移位更靠近SV。我们提出 近端钉连接到CaV通道，因为CaV通道离突触标签越近， 当脉冲导致通道打开并允许Ca 2+流入胞质溶胶时， 突触结合蛋白暴露的Ca 2+浓度越高，它触发膜结合的可能性就越大。 核聚变本文提出的研究目的是将CaV和KCa通道定位在活动区， 青蛙神经肌肉接头具有足够的分辨率，以确定它们是否与 连接到跨越PM的大分子，如果是，则确定每个通道的哪一行 是集中的。为了实现这一目标，一种涉及CaV和KCa的组织化学标记的创新方法， 将使用通道和定量电子层析成像。