A key mechanism underlying presynaptic G protein-mediated inhibition of neurotransmitter release is the interaction between G protein βγ (Gβγ) subunits and voltage-dependent Ca2+ channels. To determine the functional relevance of two of the identified Gβγ interaction sites [1], we have investigated the effects of an amino terminal (CaV2.2 [45-55]) ‘NT peptide’ and a I-II loop alpha interaction domain (CaV2.2 [377-393]) ‘AID peptide’ on G protein modulation of Ca2+ channels using whole-cell patch clamp in isolated rat superior cervical ganglion neurons (SCGNs). We also tested the effect of these peptides on synaptic transmission (at 0.1 Hz) in SCGNs long-term culture [2]. Under control conditions 10 μM of noradrenaline (NA) caused an IBa inhibition of 55.4±4.8 % (at -10 mV, n=11) and a depolarizing shift in voltage-dependence of activation (V0.5) of ~10 mV. Prepulse facilitation ratio after NA application was 1.9±0.2 (at -10 mV, n=11) and τreinhibition was 118±11 ms (at -70 mV, n=8). Under equivalent conditions, NT and AID peptides (both 1 mM, added intracellularly via the patch electrode) disrupted G protein modulation. NA-induced inhibition was significantly reduced by the NT (32.4±3.5 %, n=11) and AID (35.6±2.8 %, n=9) peptides (both P<0.05 vs control NA inhibition, Mann Whitney U-tests) and no depolarizing shift in V0.5 was observed. NA-mediated inhibition was no longer reversed by depolarizing prepulses: facilitation ratios remained unchanged for NT (1.2±0.03, n=11) and AID (1.1±0.1, n=9) peptides. Moreover, τreinhibition was significantly decreased by the NT (56±4 ms, n=5) and AID (48±8 ms, n=7) peptides (both P< 0.05 vs control, Mann WhitneyU-tests). However, a mutant AID peptide containing a modification in the proposed (QxxER) consensus sequence for G protein modulation was still able to disrupt NA effects. Presynaptic injection of the NT or the AID peptide in SCGNs in long-term culture caused an inhibition in synaptic transmission. Thus, decreases in EPSP amplitude were seen for 1 mM NT (-21±5.5 %, n=6) and 1 mM AID (-37±11 %, n=6) peptides (P<0.05 vs a control or a scrambled peptide, Student t-test). Presynaptic injection of the mutant NT peptide (1 mM) had no effect on EPSP amplitude, whilst mutant AID (1 mM) was still effective (-18±3.0 %, n=6, P< 0.05 vs a mutant NTpeptide, Student t-test). Overall, these data suggest that the CaV2.2 amino terminal and I-II loop, previously shown to interact with Gβγ subunits, contribute molecular determinants involved in modulation of neurotransmitter release. These data are also consistent with residues implicated as important in G protein modulation on the NT, but not those on the AID region, playing a major role. Small molecules based on Gβγ/Ca2+ channel interaction sites may thus have therapeutic potential in aberrant signaling pathways involving CaV2.2 channels, such as in nociception.