Molecular determinants of inactivation and G protein modulation in the intracellular loop connecting domains I and II of the calcium channel alpha1A subunit.

Synaptic transmission is regulated by G protein-coupled receptors whose activation releases G protein betagamma subunits that modulate presynaptic Ca2+ channels. The sequence motif QXXER has been proposed to be involved in the interaction between G protein betagamma 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 alpha1A subunits, which are modulated by G proteins, but not in alpha1C subunits, which are not modulated. Peptides containing the QXXER motif from adenylate cyclase 2 or from alpha1A block G protein modulation but a mutant peptide containing the sequence AXXAA does not, suggesting that the QXXER-containing peptide from alpha1A can competitively inhibit Gbetagamma modulation. Conversion of the R in the QQIER sequence of alpha1A to E as in alpha1C 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 alpha1C to R has opposite effects on voltage-dependent inactivation, although the changes are not as large as those for alpha1A. Mutation of the QQIER sequence in alpha1A to QQIEE enhanced G protein modulation, and mutation to QQLEE as in alpha1C 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 Gbetagamma. 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.