My laboratory has maintained a longstanding collaboration with Heidi Hamm's laboratory at Vanderbilt University Medical Center 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.
Briefly, this interaction was first discoved by Trillium Blackmer (Blackmer et al 2001 Science 292, 293-297) and confirmed by a key finding of its sensitivity to Botulinum A toxin cleavage of SNAP-25 by Tatyana Gerachenko (Gerachshenko et al 2005 Nature Neuroscience 8, pages 597–605). We have continued to work on this mechanism first confirming its existence in the mammalian CNS (Hamid et al 2014 J. Neuroscience 34, 260-274) and lead by Zack Zurawski at Vanderbilt, but now here in Chicago, we now have a model mouse that largely lacks this interaction (Zurawski et al 2019 Science Signaling 12, DOI: 10.1126/scisignal.aat8595).
The schematic above shows the competitive interaction between Gβγ and synaptotagmin at the SNARE complex in presynaptic terminals. Gβγ interferes with synaptotagmin binding at the C-terminal region of SNAP25.
|Simon T Alford, PhD|
Sweeney Professor of Basic Sciences and Head, Anatomy and Cell Biology Dept.
|Zack Zurawski, PhD|
|Shelagh Rodriguez, BS|
|Emily Church, BS|
|Edaeni Hamid PhD|
Edaeni graduated in 2011. She is now with Inscopix in San Fransisco
|Tatyana Gerachshenko, MD/PhD|
Tayana graduated in 2005. She is now a practising physician specializing in rehabilitation medicine
|Trillium Blackmer PhD|
Trillium graduated in 2000. She is a Senior Scientist at Thermofisher
Effects of Gβγ on vesicle fusion
GPCRs modify neurotransmission at all synapse. They have been shown to act by membrane delimited mechanisms of Gβγ either at Ca2+ channels or at the SNARE complex.
At CA1-subicular presynapses, 5-HT1B and GABAB receptors colocalize. GABABRs inhibit Ca2+ entry, whereas 5-HT1BRs Ca2+-dependently target SNARE complexes. We have demonstrated that GABABRs, alter Pr, but 5-HT1BRs reduce cleft glutamate concentrations allowing strong inhibition of AMPA- but not NMDA-receptor responses. Simulations of glutamate release and receptor binding demonstrates that experimental effects on release and low affinity antagonism are well-fit by reduced release rates. These simulations are freely available (panel to right). Train-dependent presynaptic Ca2+ accumulation forces frequency-dependent recovery of neurotransmission during 5-HT1BR activation, consistent with competition between Ca2+-synaptotagmin and Gβγ at SNARE complexes. Thus, stimulus trains in 5-HT unveil dynamic synaptic modulation and a sophisticated hippocampal output filter, that itself is modulated by colocalized GABABRs which alter presynaptic Ca2+ allowing complex presynaptic integration.
Gβγ interactions with SNARE complexes
We have determined that Gβγ interacts with the c-terminus of SNAP-25 on primed vesicle complexes to inhibit neurotransmitter release. We have developed substantial evidence that this interaction interferes with synaptotagmin 1 binding to the SNARE complex. Furthermore, during either an experimental increase in Ca2+ or during repetitive stimulation when residual Ca2+ accumulates in the presynaptic terminal this interaction is lost. However, Ca2+ imaging and simulation results indicate that this loss of interaction is not principally caused by a Ca2+ dependent loss of synaptotagmin 1- SNARE binding, but is rather mediated by another Ca2+ sensor. This question is driving our present research direction.
Simulations of glutamate release from vesicle fusion pores and subsequent activation of postsynaptic receptors
MCell simulation and parameter files
Each folder contains checkpointed sequences that can be run
with the enclosed python script (seed_mdl).
This link downloads a zip file containing a number of mdl files and a python script. The python script (seed_mdl.py) calls a sequence of .mdl files that should be in the current terminal (or cmd) directory. These files (d4.mdl to Full.mdl) call a sequence of expanding pore size geometries for different durations. The scripts calls mcell provided mcell is in the path. Mcell is available at mcell.org where instructions for its download and use are available. Note the 3D meshes were generated in Blender 2.79 and the models were run in mcell 3.
Methods for downloading and running these simulations is the same as Full fusion (above). These simulations arrest at particular pore diameters
This models 3 vesicles fusing
|In this model, receptors on the postsynaptic terminal are constrained to particular locations|