5HT Modulates the Mode of Fusion through the Interaction of Gbg, Calcium-synaptotagmin and SNARE Proteins

Fast synchronous neurotransmission occurs when an action potential triggers Ca2+ influx through voltage-gated calcium channels. The creation of a full fusion pore is inhibited by the G subunit of a Gi/o-coupled serotonin receptor, favoring kiss-and run fusion. The inhibition of G involves competition in a Ca2+-dependent manner with Synaptotagmin binding of SNAP-25 at the C-terminal. Using a novel approach to inject mutant SNAP-25 into axons we discovered two mutant SNAP-25 proteins (8A and 3) which showed decreased affinity for G, rescued synaptic transmission but failed to participate in 5-HT mediated inhibition. Inhibition of G is relieved under high Ca2+ transients. Diffusional rates and endogenous buffering of Ca2+ suggests that the hyperlocal Ca2+ concentration after a given action potential is crucially important for determining Ca2+ dependent interactions between Synaptotagmin, G and the ternary SNARE complex. This makes the open probability of VGCCs in response to a single stimulus as well as during high frequency stimulation relevant. Utilizing the newly constructed Lattice Light Sheet Microscopy I have shown that Ca2+ entry at a given active zone is variable, stochastic and independent between active zones. I propose that this variability is due to VGCCs opening in cassettes, with subsequent stimulation causing inactivation or facilitation of a subset of VGCCs.