Molecular determinants of inactivation and G protein modulation in the intracellular loop connecting domains I and II of the calcium channel α(1A) subunit

Synaptic transmission is regulated by G protein-coupled receptors whose activation releases G protein βγ subunits that modulate presynaptic Ca(2+) channels. The sequence motif QXXER has been proposed to be involved in the interaction between G protein βγ 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 Ca(2+) channel α(1A) subunits, which are modulated by G proteins, but not in α(1C) subunits, which are not modulated. Peptides containing the QXXER motif from adenylate cyclase 2 or from α(1A) block G protein modulation but a mutant peptide containing the sequence AXXAA does not, suggesting that the QXXER-containing peptide from α(1A) can competitively inhibit Gβγ modulation. Conversion of the R in the QQIER sequence of α(1A) to E as in α(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 α(1C) to R has opposite effects on voltage-dependent inactivation, although the changes are not as large as those for α(1A). Mutation of the QQIER sequence in α(1A) to QQIEE enhanced G protein modulation, and mutation to QQLEE as in α(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 Gβγ. Overlap of this motif with the consensus sequence for binding of Ca(2+) channel β subunits suggests that this region of L(I-II) is important for three different modulatory influences on Ca(2+) channel activity.