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
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Synaptic impulse transmission fundamentally relies on the coupling of neuron impulses with neurotransmittersecretion 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 â€˜Active Zone Materialâ€™ (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. SVsheld 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 anelectrical impulse at an active zone causes voltage-gated Ca2+ (CaV) channels to open and allow Ca2+ to enterthe 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 membranefusion 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 channelsto docked SVs and KCa channels strongly influences impulse-secretion coupling. In axon terminals of a modelsynapse, 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 quantitativelystudy the 3D macromolecular structure of AZM at frog neuromuscular junctions and found that the members ofa 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 arriveswere associated with pegs in the row proximal to the SVs that were displaced closer to them. We proposedthat 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 theconcentration of Ca2+ exposure to synaptotagmin and the greater the probability that it will trigger membranefusion. The objective of the research proposed here is to localize the CaV and KCa channels at active zones offrog neuromuscular junctions with sufficient resolution to determine if they are associated with the pegs thatare connected to the macromolecules that span the PM, and if they are, to determine which row each channelis concentrated. To meet this objective, an innovative method involving histochemical labeling of CaV and KCachannels together with quantitative electron tomography will be used.