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.

The modulation of presynaptic calcium channel activity by second messengers provides a fine tuning mechanism for neurotransmitter release. In neurons, the activation of certain G protein-coupled receptors reduces N-type channel activity by ϳ60%. In contrast, activation of protein kinase C (PKC) results in an approximately 50% increase in N-type channel activity, and subsequent G protein inhibition is antagonized. Here, we describe the molecular determinants that control the dual effects of PKC-dependent phosphorylation. Calcium influx through neuronal voltage-dependent calcium channels mediates a range of cytoplasmic responses, such as neurotransmitter release, proliferation, and the activation of calcium-dependent enzymes. Most neurons express multiple calcium channel types with distinct functional properties, and molecular cloning has identified genes encoding at least eight different neuronal calcium channel ␣ 1 subunits (termed ␣ 1A through ␣ 1H ). Functional expression in heterologous expression systems has revealed that ␣ 1A encodes for P/Q-type calcium channels (1-3); ␣ 1B defines an -conotoxin GVIA-sensitive Ntype channel (4 -6); ␣ 1C , ␣ 1D and ␣ 1F are L-type calcium channels (7-9); and ␣ 1E is a unique calcium channel with properties common to both high threshold and low threshold calcium channels (10, 11). More recently, ␣ 1G and ␣ 1H have been shown to encode members of the family of T-type calcium channels (12). Among the eight types of ␣ 1 subunits, ␣ 1A and ␣ 1B are predominantly located at more distal dendritic and presynaptic nerve terminals (13,14) and are directly coupled to the presynaptic vesicle release machinery (15, 16).The physiological properties of presynaptic calcium channels are extensively modulated by second messenger molecules, including protein kinase C and G protein ␤␥ subunits (17-21). The activation of certain G protein-coupled seven-helix transmembrane receptors mediates a pronounced voltage-dependent inhibition of both N-type and P/Q-type calcium currents (Refs. 22-25; for a review, see Ref. 26). This inhibition is probably caused by direct 1:1 binding of G protein ␤␥ subunits to the calcium channel ␣ 1 subunit, resulting in a reluctance of the channels to undergo opening in response to membrane depolarization (27, 28). In contrast, stimulation of protein kinase C-dependent phosphorylation results in a substantial up-regulation of N-type channel activity (19). PKC 1 and G ␤␥ modulation are functionally coupled (termed cross-talk), such that PKC-dependent phosphorylation of the channel antagonizes subsequent G protein inhibition (20,21,29).We have previously shown that cross-talk between G protein and PKC pathways mainly occurs at the level of the calcium channel ␣ 1 subunit (29). In particular, the cytoplasmic linker connecting domains I and II of the ␣ 1B subunit is a crucial determinant of both G protein inhibition and PKC-G protein cross-talk. This region has also been implicated in PKC-dependent up-regulation of N-type calcium channels (19), suggesting the possibility o...

In mRNA samples isolated from a gastrin (G) cell-enriched human antral cell preparation, reverse transcription-polymerase chain reaction identified products encoding part of the alpha 1-subunit of class C and D L-type voltage-dependent Ca2+ channels (VDCCs). Analysis of the polymerase chain reaction products demonstrated a 100% homology with the known human gene sequences. An antibody to the class D alpha 1-subunit immunostained 30-40% of the cultured cells; of these 90% were gastrin immunoreactive. Gastrin release stimulated by terbutaline (beta 2-agonist) and forskolin was abolished by blockade of L-type VDCCs; the effect of 3.6 mM extracellular Ca2+ was only partially reversed. In G cells the rise in intracellular Ca2+ observed in response to increasing extracellular Ca2+ from 0.5 to 3.6 mM was reduced by nitrendipine. These results indicated that human antral cells expressed class C and D L-type VDCCs. Activation of G cells with beta-adrenergic agonists required an influx of extracellular Ca2+ through these channels to stimulate gastrin release. However, activation of L-type channels was not the only mechanism underlying Ca(2+)-stimulated gastrin release.