There is growing evidence that Gbg inhibits synaptic transmission by modulation downstream of Ca2+ entry. That G proteins directly target the release apparatus was first demonstrated in the neuromuscular junction (Silinsky, 1984). Spontaneous exocytotic events, allow isolation and study of exocytotic modulatory processes that occur independently of Ca2+ entry. The frequency of these events are regulated by GPCRs (Scanziani et al, 1995).

In support of the hypothesis that G proteins inhibit vesicle fusion directly, G proteins inhibit exocytosis after cell permeabilization, suggesting a role late in the exocytotic event (Luini and De Matteis, 1990). Additionally, exocytotic processes in pancreatic b cells, peritoneal mast cells, chromaffin cells, PC12 cells and secretory granules are regulated independently of Ca2+ entry by G proteins (Lang, 1999). Gbg interacts directly with the fusion machinery in rat mast cells (Pinxteren et al, 1998). In the lamprey giant synapse, 5-HT-mediated inhibition (Fig 1A) does not reduce presynaptic Ca2+ entry (Takahashi et al, 2001). Furthermore, the actions of 5-HT at a GPCR are abolished by intracellular block of activated Gbg (Blackmer et al, 2001). A mechanism for a direct interaction between Gbg and the core vesicle fusion machinery was suggested by the finding that Gbg which inhibits exocytosis (Fig 1B) directly binds SNARE proteins syntaxin and SNAP-25 (Jarvis et al, 2000; Blackmer et al, 2001) as well as the cysteine string protein (CSP) (Magga et al, 2000). It is now clear that Gbg can inhibit release by a direct interaction with the SNARE complex (Blackmer et al, 2005). Indeed, it is apparent that this interaction can occur across a wide range of models of exocytosis, from amygdala synapses (Delaney et al, 2007) to endocrine cells (Blackmer et al, 2005) to spinal synapses (Gerachshenko et al, 2005; Yoon et al, 2007). However, it is also clear that this modification represents a different mechanism than that identified by effects on mini frequency, because mini frequency is unaffected by this mechanism (Delaney et al, 2007; Schwartz et al, 2007). This interaction occurs at a late phase after priming in vesicles whose SNARE complex is formed and not available for cleavage by botulinum toxin B (Gerachshenko et al, 2005). Furthermore, Gbg binds to the C-terminal regions of the formed SNARE complex and competes with Ca2+-dependent synaptotagmin binding to the SNARE complex (Yoon et al, 2007). In this way a Ca2+ dependency is conferred on Gbg mediated presynaptic inhibition because high presynaptic Ca2+ concentrations allow synaptotagmin to compete more effectively (Fig 1).

Figure 1 model for the target of Gbg on the SNARE complex.
A. 5-HT inhibits EPSCs evoked from reticulospinal axons and recorded in ventral horn neurons. In some, the EPSC comprises early electrical component that is not inhibited and a later component blocked by 5-HT.
B. Gbg injected into the presynaptic terminal also inhibits these EPSCs.
C. Presynaptic GPCR activation liberates Gbg into the presynaptic terminal which competes with the Ca2+ sensor, synaptotagmin for binding to the C terminal region of the ternary SNARE complex.

This direct modification of synaptotagmin-SNARE complex interactions is likely to be profoundly altered by the state of the synapse, a state that may be affected by other activated presynaptic receptors, and by recent activity (Trussell, 2002; Zucker and Regehr, 2002; Awatramani et al, 2005). These effects cause varying concentrations of residual presynaptic Ca2+ during stimulus trains or heterosynaptic input to the presynaptic terminal. GPCRs inhibit exocytosis by a direct action of Gbg on the C-terminal region SNARE complex (Blackmer et al, 2001; Blackmer et al, 2005; Gerachshenko et al, 2005). Gbg competes with Ca2+-dependent synaptotagmin binding at this region to inhibit exocytosis (Yoon et al, 2007). In turn, raised presynaptic Ca2+ concentrations prevent Gbg-mediated inhibition (Figs. 16,17). Thus, during stimulus trains, which are likely to be much more reflective of in situ synaptic function than single stimuli, 5-HT-mediated inhibition is modulated by rising presynaptic [Ca2+]. We hypothesize that the effect of GPCRs that directly target the release apparatus is profoundly modified by presynaptic activity that alters Ca2+ concentrations. The implications for this may be profound. At the physiological level, activity trains may inactivate presynaptic inhibitory mechanisms reinforcing rhythmic output during the train, but allowing depression of output at lower frequencies of firing between bursts. At the systems level, for example, we observe that receptors that reinforce Ca2+ entry to presynaptic terminals during stimulus trains (Cochilla and Alford, 1998) are necessary to sustain spinal motor output (Takahashi and Alford, 2002). At many central synapses, release of Ca2+ from internal stores driven either by voltage gated Ca2+ entry or by receptor activation can modify neurotransmitter release. Similarly, 5-HT application to the spinal cord increases duration of trains of motor output as well as interburst intervals (Schwartz et al, 2005). This phenomenon also opens the possibility for synergistic effects of Gbg at the synapse. If Gbg were to both reduce Ca2+ entry (Mizutani et al, 2006), and compete with Ca2+-dependent synaptotagmin binding to the SNARE complex (Yoon et al, 2007), then effects of the activating receptor (eg 5-HT1B) will be much more powerful. We will investigate the role that Ca2+ plays in modifying the functional outcome of presynaptic GPCR activation on synaptic transmission in physiologically relevant paradigms. The importance of a convergence of competing effects of Ca2+-synaptotagmin and Gbg is even more clearly delineated by recent work from our laboratory and others demonstrating that Gbg causes kiss and run fusion (Chen et al, 2005; Photowala et al, 2006; Schwartz et al, 2007) while high frequency stimulation and high presynaptic Ca2+ concentrations favor full fusion (Harata et al, 2006a). Complex interactions between the fusion machinery, G proteins and presynaptic Ca2+ entry clearly may lead to qualitative as well as quantitative changes in neurotransmission.