Synaptic transmission is regulated by G protein-coupled receptors whose activation releases G protein beta gamma subunits that modulate presynaptic Ca2+ channels, The sequence motif QXXER has been proposed to be involved in the interaction between G protein beta gamma subunits and target proteins including adenylyl cyclase 2. This motif is present in the intracellular loop connecting domains I and II (L(I-II)) of Ca2+ channel alpha(1A) subunits, which are modulated by G proteins, but not in alpha(1C) subunits, which are not modulated, Peptides containing the QXXER motif from adenylate cyclase 2 or from alpha(1A) block G protein modulation but a mutant peptide containing the sequence AXXAA does not, suggesting that the QXXER-containing peptide from alpha(1A) can competitively inhibit G beta gamma modulation, Conversion of the R in the QQIER sequence of alpha(1A) to E as in alpha(1C) slows channel inactivation and shifts the voltage dependence of steady-state inactivation to more positive membrane potentials, Conversion of the final E in the QQLEE sequence of alpha(1C) to R has opposite effects on voltage-dependent inactivation, although the changes are not as large as those for alpha(1A). Mutation of the QQIER sequence in alpha(1A) to QQIEE enhanced G protein modulation, and mutation to QQLEE as in alpha(1C) greatly reduced G protein modulation and increased the rate of reversal of G protein effects, These results indicate that the QXXER motif in L(I-II) is an important determinant of both voltage-dependent inactivation and G protein modulation, and that the amino acid in the third position of this motif has an unexpectedly large influence on modulation by G beta gamma. Overlap of this motif with the consensus sequence for binding of Ca2+ channel beta subunits suggests that this region of L(I-II) is important for three different modulatory influences on Ca2+ channel activity.