Background:
Neurotransmitter release from mouse motor nerve terminals occurs at highly organized structures called active zones (AZs), where P/Q-type voltage-gated calcium channels (VGCCs), structural proteins, and docked synaptic vesicles govern neurotransmitter release during single action potentials. Understanding AZ organization is important not only in healthy synapses, but also for neuromuscular diseases that disrupt the protein content and/or organization at the neuromuscular junction (NMJ). Despite decades of research on the NMJ, little is known of the identity and organization of AZ proteins. To better understand the pathophysiology of diseases that target the NMJ, we need to determine the density and distribution of AZ proteins. Of particular importance are the VGCCs that provide the calcium ions necessary for neurotransmitter release. Prior electron microscopy studies have shown that AZs are composed of two double rows of trans-membrane proteins, a fraction of which are presumed to be presynaptic VGCCs. However, the number of VGCCs, and their location within the double rows, remains unknown.
Objectives:
To determine the number and location of VGCCs within the AZ, we utilized DNA PAINT super-resolution microscopy. Additionally, to estimate the number of VGCCs that contribute calcium ions to each synaptic vesicle fusion event, we performed presynaptic calcium imaging and intracellular recordings in various concentrations of the VGCC blocker ω-Agatoxin IVA to determine the VGCC-release site cooperativity at the mammalian NMJ.
Results:
Using DNA PAINT microscopy, we found that there are a small number of P/Q-type VGCCs (~1-6) per AZ. Our VGCC-release site cooperativity experiments further support this finding by suggesting that most calcium ions contributing to a release event originate from only ~1-2 VGCCs, which is expected with few VGCCs per AZ that each have a low probability of opening during an action potential.
Conclusions:
Our data suggest that each synaptic vesicle fusion event at the mouse NMJ is triggered mostly by the calcium ions from a single open VGCC. These data lead to the conclusion that at the mouse NMJ there are very few presynaptic VGCCs per AZ and there is nanodomain coupling between VGCCs and transmitter release. These findings will help inform future interpretations of the changes in AZ protein content and organization that occur in diseases affecting the NMJ, such as Lambert-Eaton Myasthenic Syndrome.