Our laboratory has maintained a longstanding collaboration with Heidi Hamm's laboratory at Vanderbilt University Medical Center aiming to understand how GPCRs and G proteins modulate neurotransmitter release. We discovered a direct mechanism by which Gβγ interacts with the SNARE complex and have characterized this interaction in detail over the last few years. 

Activation of presynaptic Gi/o-coupled receptors by hormones, neurotransmitters (NT) and neuromodulators leads to decreased neurotransmission. This decreased release provides an important control mechanism for autoreceptors to guard against over-activation, and an important homeostatic mechanism. For heteroreceptors, it is a critical component of synaptic integration mediating circuitry-level effects. Fast membrane-delimited inhibition of secretion may occur via Gβγ regulation of voltage-dependent Ca2+channels (VDCCs). However, a direct interaction between Gβγ and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins also leads to inhibition of exocytosis downstream of Ca2+ entry. This mechanism is not only more acute and direct in controlling evoked release, leaving secondary effects of presynaptic Ca2+ unaffected, but is also able to modify components of exocytosis not available to mechanisms that control release probability. These include modifying the concentration of neurotransmitter released by interacting with a region of the SNARE complex that controls fusion rate, but also modifying spontaneous release, which has important roles in its own right. The same synapses can have different Gi/o-GPCR-triggered modulation of neurotransmitter release by different mechanisms. For example, in hippocampal neurons, GABAB receptors cause decreased Ca2+ entry and 5HT1b receptors inhibit exocytosis by directly acting on SNAREs at the same synapse: this allows for presynaptic neural integration. What could be the mechanistic basis of this specificity? There is considerable evidence that unique Gβγ isoforms play specific roles in mediating interactions with both receptors and effectors. Our recent in vivo proteomic studies of Gβγ specificity suggest that it might come from receptor selection of particular Gβγ subunits, and the affinity of those Gβγ‘s for the SNARE complex (unpublished).

This interaction was first discoved by Trillium Blackmer in our laboratories.

Blackmer, T., Larsen, E., Takahashi, M., Martin, T. F. J., Alford, S. & Hamm, H. E. (2001) Heterotrimeric G protein Gβγ subunits mediate presynaptic inhibition by regulating exocytotic fusion downstream of Ca2+ entry. Science, 292:293-297

This was confirmed by a key finding of its sensitivity to Botulinum A toxin cleavage of SNAP-25 by Tatyana Gerachenko working in the Alford laboratory using lamprey giant reticulospinal axons along with Trillium Blackmer showing similar effects in PC12 cell models. Loss of the c-terminus of SNAP25 weakens the ability of Gβγ to interact with the SNARE complex late in the exocytotic event to inhibit synaptic release. 

Gerachshenko T, Blackmer T, Yoon EJ, Bartleson C, Hamm HE, Alford S. (2005)  Gbg acts at the C terminus of SNAP-25 to mediate presynaptic inhibition. Nature Neuroscience 8: 597-605.

Blackmer T, Larsen EC, Bartleson C, Kowalchyk JA, Yoon E-J, Preininger AM, Alford S, Hamm HE, Martin TFJ. (2005) G protein  directly regulates SNARE protein fusion machinery for secretory granule exocytosis. Nature Neuroscience 8: 421-425 

This work's importance was discussed in a News and Views article by Jane Sullivan in Nature Neuroscience.

We have continued to work on this mechanism first confirming its existence in the mammalian CNS. Edaeni Hamid demonstrated this and contrasted the  effects of direct Gβγ SNARE interactions with the less direct mechanism by which Gβγ interacts with presynaptic calcium channels to reduce calcium entry and therefore neurotransmitter release. 

E Hamid, E Church, CA Wells, Z Zurawski, HE Hamm, S Alford (2014) Modulation of neurotransmission by GPCRs is dependent upon the microarchitecture of the primed vesicle complex. Journal of Neuroscience 34 (1), 260-274

E Church, E Hamid, Z Zurawski, M Potcoava, E Flores-Barrera, Synaptic integration of subquantal neurotransmission by colocalized G protein-coupled receptors in presynaptic terminals Journal of Neuroscience 42 (6), 980-1000

We determined residues on the SNARE complex protein, SNAP25, that are especially important for the Gbg-SNARE interaction. From this work lead by Zack Zurawski, first at Vanderbilt, but now here in Chicago, we have developed  a model mouse that largely lacks this interaction. This model shows a number of defects both central and peripheral. 

Zurawski Z, AD Thompson Gray, Brady LJ, Page B, Church E, Harris NA, Dohn MR, Yim YY, Hyde K, Mortlock DP, Jones CK, Winder DG, Alford S, Hamm HE. (2019) Disabling the Gβγ-SNARE interaction disrupts GPCR-mediated presynaptic inhibition, leading to physiological and behavioral phenotypes. Science signaling 12 (569), eaat8595

RP. Ceddia, Z Zurawski, A Thompson Gray, F Adegboye, A McDonald-Boyer, F Shi, D Liu, J Maldonado, J Feng, Y Li, S Alford, JE. Ayala, OP. McGuinness, S Collins, HE. Hamm (2023). Removing the Gβγ-SNAP25 brake on exocytosis enhances insulin action, promotes adipocyte browning, and protects against diet-induced obesity. Journal of Clinical Investigation (In press).