Figure 1.

GB_1aRs, Gα_oβ₁γ₂ G-protein subunits, and Ca_V2.2 channels are precoupled at single hippocampal boutons. A, Representative confocal images of pyramidal neuron axons in hippocampal cultures that were cotransfected with GB_1a^YFP/Ca_V2.2^CFP, GB_1a^YFP/Gα_o^CFP, GB_1a^CFP/Gβ₁^YFP and Ca_V2.2^CFP/Gβ₁^YFP. Scale bars, 2 μm. B, FRET was detected between GB_1a^YFP/Ca_V2.2^CFP (n = 33, N = 7), GB_1a^YFP/Gα_o*^94CFP (n = 53), GB_1a^CFP/Gβ₁^YFP (n = 45, N = 10), GB_1a^CFP/Gγ₂^YFP (n = 21, N = 4), Ca_V2.2^CFP/Gβ₁^YFP (n = 31, N = 6) proteins under miniature synaptic activity at single hippocampal boutons. To verify FRET specificity, E was measured between the CFP-tagged proteins of interest and nonrelated TNFR₂^YFP. Error bars indicate SEM. ***p < 0.001. C, FRET efficiency is plotted for individual presynaptic boutons as function of CFP/YFP intensity ratio (F_CFP/F_YFP). No correlation was found: Spearman r is 0.14, 0.11, −0.08, and 0.05 for GB_1a^YFP/Ca_V2.2^CFP, GB_1a^YFP/Gα_o*^94CFP, GB_1a^CFP/Gβ₁^YFP, and Ca_V2.2^CFP/Gβ₁^YFP, respectively (p > 0.5). D, FRET was detected between GB_1a^YFP/Ca_V2.2^CFP (n = 8, N = 3), GB_1a^YFP/Gα_o^CFP (n = 9, N = 3), GB_1a^CFP/Gβ₁^YFP (n = 8, N = 3), and Ca_V2.2^CFP/Gβ₁^YFP (n = 12, N = 4) proteins in nonreleasing TeTx-pretreated hippocampal boutons. E, FRET was detected between GB_1a^YFP/Ca_V2.2^CFP (n = 9, N = 3), GB_1a^YFP/Gα_o^CFP (n = 9, N = 3), GB_1a^CFP/Gβ₁^YFP (n = 10, N = 3), and Ca_V2.2^CFP/Gβ₁^YFP (n = 9, N = 3) proteins in nonreleasing immature (4–5 DIV) hippocampal neurons.