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

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 轴突终末沿突触前质膜(PM)的特殊部位分泌的Ac- 动态区。所有突触的活动区都有类似的细胞器，称为“活动区物质”(AZM)， 它们由同源蛋白组成，它们组装成不同类别的Azm大分子； AZM调节导致停靠的突触小泡(即SVS)分泌神经递质的事件 与首相保持联系)。确定组成AZM的蛋白质的身份是必要的- 很难理解支配调节神经递质Se-2的分子机制的一般规则。 在正常、实验和疾病条件下，整个神经系统的排泄。一位新成员的到来 活动区的电脉冲导致电压门控钙离子(Cav)通道开放并允许钙离子进入 导致通道口附近钙离子浓度升高的胞浆，持续时间很短。 时间太短了。如果足够浓度的钙离子与SV蛋白突触素相互作用，它就会触发细胞膜 融合和神经递质分泌，这是所描述的冲动-分泌耦合的定义阶段。 进入胞浆的Ca~(2+)也激活Ca~(2+)门控K~+(KCA)通道，使质膜重新极化和去激活。 激活CAV通道以阻止进一步的神经递质分泌。因此，CAV通道的相对近似性 对接的SVS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通道都是复合性的。 以平行的双排阵列排列的横跨PM的活性区域的大分子的Nent。 在冷冻断裂的复制品中刻画。我们实验室以前的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的三维大分子结构，发现 一类特殊的名为pegs的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，停靠的SVS与PM融合的可能性最大 与SVS近侧排中移位较近的钉子有关。我们向他求婚 近端的钉子连接到CAV通道是因为CAV通道离突触标记越近- 分钟当脉冲导致通道打开并允许钙离子流入胞浆时， 突触素作用下的钙离子浓度越高，触发膜的概率越大 核聚变。这项研究的目的是将Cav和Kca通道定位于 具有足够分辨率的青蛙神经肌肉接头，以确定它们是否与 连接到横跨PM的大分子，如果是，则确定每个通道的哪一行 是集中的。为了达到这一目标，一种创新的方法包括CAV和KCA的组织化学标记 将使用通道和定量电子断层扫描。

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.