Direct interactions between the presynaptic N type calcium channel and the subunit of the heterotrimeric G protein complex cause voltage dependent inhibition of N type channel activity, crucially influencing neurotransmitter release and contributing to analgesia caused by opioid drugs. Previous work using chimeras of the G protein subtypes G₁ and G₅ identified two 20-amino acid stretches of structurally contiguous residues on the G₁ subunit as critical for inhibition of the N type channel (Doering et al., J. Biol. Chem. 279, 29709-29717). To identify key modulation determinants within these two structural regions, we performed scanning mutagenesis in which individual residues of the G₁ subunit were replaced by corresponding G₅ residues. Our results show that G₁ residue Ser¹⁸⁹ is critical for N type calcium channel modulation, whereas none of the other G₁ mutations caused statistically significant effects on the ability of G₁ to inhibit N type channels. Structural modeling shows residue 189 is surface exposed, consistent with the idea that it may form a direct contact with the N type calcium channel 1 subunit during binding interactions.