Identification of an integration center for cross-talk between protein kinase C and G protein modulation of N-type calcium channels

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 similar to 60%. 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. The double substitution of two adjacent PKC consensus sites in the calcium channel domain I-II linker (Thr(422), Ser(425)) to alanines abolished both PKC-dependent up-regulation and the PKC-G protein cross-talk. The single substitution of Ser(425) to glutamic acid abolished PRC up-regulation but had no effect on G protein modulation. Replacement of Thr(422) With glutamic acid eliminated PKC-dependent up-regulation and mimicked the effects of PKC phosphorylation on G protein inhibition. Our data suggest that Thr(422) mediates the antagonistic effect of PKC on G protein modulation, while phosphorylation of either Thr(422) or Ser(425) are sufficient to increase N-type channel activity. Thus, Thr(422) serves as a molecular switch by which PKC is able to simultaneously trigger the upregulation of channel activity and antagonize G protein inhibition.