Abstract

Presynaptic inhibition via G-protein-coupled receptors (GPCRs) and voltage-gated Ca²⁺ channels constitutes a widespread regulatory mechanism of synaptic strength. Yet, the mechanism of intermolecular coupling underlying GPCR-mediated signaling at central synapses remains unresolved. Using FRET spectroscopy, we provide evidence for formation of spatially restricted (<100 Å) complexes between GABA_B receptors composed of GB_1a/GB₂ subunits, Gα_oβ₁γ₂ G-protein heterotrimer, and Ca_V2.2 channels in hippocampal boutons. GABA release was not required for the assembly but for structural reorganization of the precoupled complex. Unexpectedly, GB_1a deletion disrupted intermolecular associations within the complex. The GB_1a proximal C-terminal domain was essential for association of the receptor, Ca_V2.2 and Gβγ, but was dispensable for agonist-induced receptor activation and cAMP inhibition. Functionally, boutons lacking this complex-formation domain displayed impaired presynaptic inhibition of Ca²⁺ transients and synaptic vesicle release. Thus, compartmentalization of the GABA_B1a receptor, Gβγ, and Ca_V2.2 channel in a signaling complex is required for presynaptic inhibition at hippocampal synapses.