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.

The Ca2+ that enters an axon terminal through voltage-gated Ca2+ channels in response to an electrical im- pulse triggers neurotransmitter secretion and activates nearby Ca2+-gated K+ channels to repolarize the mem- brane and cease further secretion. The experiments in this application aim to localize the voltage-gated Ca2+ channels and Ca2+-gated K+ channels at active zones at molecular-scale resolution. The results will provide mechanistic insights of the molecular events that regulate neurotransmitter secretion, and are necessary to advance our understanding of how active zones are formed during development, how they are maintained in the adult, how they are affected as a result of disease and trauma, and how they behave during aging.