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.

突触脉冲传递从根本上依赖于神经震荡与神经递质的耦合 从轴突末端的突触前质膜（PM）沿特殊部位的分泌，称为ac- tive区。所有突触的活性区都有可比的细胞器，称为“活跃区材料”（AZM），， 这些由同源蛋白组成，这些蛋白质组装以形成不同类别的AZM大分子； AZM调节导致对接突触蔬菜（SV）的神经递质分泌的事件（即SVS 与PM接触）。确定组装形成AZM的蛋白质的身份是必需的 了解控制神经递质的分子机制的一般规则 在正常，实验和疾病状况下整个神经系统的创造。一个 电动区域的电脉冲导致电压门控Ca2+（CAV）通道打开并允许CA2+进入 胞质溶胶在通道嘴附近导致Ca2+浓度升高，以进行短暂的围裙 时间。如果足够浓度的Ca2+与SV蛋白突触抗体相互作用，则会触发膜 融合和神经递质分泌，这是描述的脉冲分泌耦合的定义阶段。 进入细胞质的Ca2+还激活了Ca2+的K+（KCA）通道，以使PM重复并脱离 Vate cav频道阻止了进一步的神经递质分泌。那，Cav通道的相对接近 停靠SV和KCA频道强烈影响冲动分泌耦合。在模型的轴突终端中 突触，青蛙神经肌肉连接，长期以来一直怀疑CAV和KCA通道都是综合的 跨越PM的大分子的nent在排列的有效区域，并行双行阵列排列 用冻结折叠式复制品进行脚本。我们实验室的先前研究使用电子断层扫描来定量 研究青蛙神经肌肉连接处AZM的3D大分子结构，发现 一种称为PEG的特定类的AZM大分子连接到跨PM的大分子。 我们还发现，当脉冲到达时，停靠的SV与PM融合的可能性最大 我们提出了钉在靠近它们的SV的行代理中。 近端PEG连接到CAV通道，因为CAV通道越接近突触 当脉冲导致通道打开并允许Ca2+进入细胞质时的最小值时， Ca2+暴露于突触量的浓度，触发膜的可能性越大 融合。这里提出的研究的目的是将CAV和KCA通道定位在 Frog神经肌肉连接处具有足够的分辨率，以确定它们是否与PEG相关联 连接到跨PM的大分子，如果是的，则可以确定每个通道 是集中的。为了满足这一目标，一种创新方法，涉及CAV和KCA的组织化学标记 通道将与定量电子断层扫描一起使用。