The modulation of voltage-dependent Ca2+ channels at presynaptic nerve terminals is an important factor in the control of neurotransmitter release and synaptic efficacy. Some terminals contain multiple Ca2(+)-channel subtypes (N and P/Q), which are differentially regulated by G-protein activation and by protein kinase C (PKC)-dependent phosphorylation. Regulation of channel activity by crosstalk between second messenger pathways has been reported although the molecular mechanisms underlying crosstalk have not been described. Here we show that crosstalk occurs at the level of the presynaptic Ca2(+)-channel complex. The alpha1 subunit domain I-II linker, which connects the first and second transmembrane domains, contributes to the PKC-dependent upregulation of channel activity, while G-protein-dependent inhibition occurs through binding of Gbetagamma to two sites in the I-II linker. Crosstalk results from the PKC-dependent phosphorylation of one of the Gbetagamma binding sites which antagonizes Gbetagamma-induced inhibition. The results provide a mechanism for the highly regulated and dynamic control of neurotransmitter release that depends on the integration of multiple presynaptic signals.