Year-end Sale % OFF 
Abstract
Gi/o-coupled G protein-coupled receptors can inhibit neurotransmitter release at synapses via multiple mechanisms. In addition to Gβγ-mediated modulation of voltage-gated calcium channels (VGCC), inhibition can also be mediated through the direct interaction of Gβγ subunits with the soluble N-ethylmaleimide attachment protein receptor (SNARE) complex of the vesicle fusion apparatus. Binding studies with soluble SNARE complexes have shown that Gβγ binds to both ternary SNARE complexes, t-SNARE heterodimers, and monomeric SNAREs, competing with synaptotagmin 1(syt1) for binding sites on t-SNARE. However, in secretory cells, Gβγ, SNAREs, and synaptotagmin interact in the lipid environment of a vesicle at the plasma membrane. To approximate this environment, we show that fluorescently labeled Gβγ interacts specifically with lipid-embedded t-SNAREs consisting of full-length syntaxin 1 and SNAP-25B at the membrane, as measured by fluorescence polarization. Fluorescently labeled syt1 undergoes competition with Gβγ for SNARE-binding sites in lipid environments. Mutant Gβγ subunits that were previously shown to be more efficacious at inhibiting Ca2+-triggered exocytotic release than wild-type Gβγ were also shown to bind SNAREs at a higher affinity than wild type in a lipid environment. These mutant Gβγ subunits were unable to inhibit VGCC currents. Specific peptides corresponding to regions on Gβ and Gγ shown to be important for the interaction disrupt the interaction in a concentration-dependent manner. In in vitro fusion assays using full-length t- and v-SNAREs embedded in liposomes, Gβγ inhibited Ca2+/synaptotagmin-dependent fusion. Together, these studies demonstrate the importance of these regions for the Gβγ-SNARE interaction and show that the target of Gβγ, downstream of VGCC, is the membrane-embedded SNARE complex.
Highlights
Gi/o-coupled G protein-coupled receptors can inhibit neurotransmitter release at synapses via multiple mechanisms
To examine binding of G␥ to full-length t-soluble N-ethylmaleimide attachment protein receptor (SNARE) complexes embedded in lipid bilayers, we developed an assay using total internal reflection (TIRF) fluorescence intensity and anisotropy (Fig. 1A)
Purified G␥ subunits fluorescently labeled at primary amine residues with Alexa Fluor 488 N-hydroxysuccinimide ester were applied from a pipette over a t-SNARE-containing bilayer on a coverslip illuminated with TIRF or epifluorescence
Summary
Previous studies of G␥–SNARE interactions used recombinant soluble SNARE complexes in aqueous solution to show that G␥ binds to ternary SNARE complexes, t-SNARE heterodimers, and the monomeric SNARE proteins SNAP25, syntaxin1A, and VAMP2 (28 –30). G␥ subunits containing Ala mutations of two residues on the G␣-binding surface of G, Lys-78 and Trp-332, inhibited exocytosis at a significantly higher potency than wild type [28] To determine if this was due to increased affinity for t-SNAREs, we tested this mutant G1␥2 subunit in the TIRF assay. Fluor 488-synaptotagmin 1 C2AB binding to t-SNARE complexes consisting of syntaxin1A and SNAP25 embedded in lipid membranes as in Fig. 2A at a Ca2ϩ concentration of 175 Ϯ 25 M. Application of Ca2ϩ increased the anisotropy of the fluorescent TIRF signal in a saturable manner produced by application of fluorescent synaptotagmin 1 to the t-SNARE containing bilayer (Fig. 2B). G1␥1 was found to inhibit fusion 1.8-fold more potently in a concentration-dependent manner at this reduced concentration of synaptotagmin 1 (Fig. 6E)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
