Interaction between Ca2+ release from inositol trisphosphate sensitive stores and Ca2+ entry through neuronal Ca2+ channels expressed in Xenopus oocyte.

Rat cerebellar RNA injected into Xenopus oocytes leads to the expression of putative P-type voltage-dependent Ca2+ channels (VDCCs). The monitoring of intracellular Ca2+ variations by recording the Ca2+ dependent chloride current in voltage clamped oocytes indicates that activation of these Ca2+ channels by depolarization gives rise to two distinct components of cytosolic Ca2+ elevation. If the early component (T1) can be directly attributed to the Ca2+ entry through VDCCs, the second delayed one (T2) is related to a Ca2+ release from InsP3 sensitive stores activated following Ca2+ entry. Modifications of cytosolic Ca2+ by direct injection of Ca2+ into oocytes or by increasing the Ca2+ influx through VDCCs suggest that the Ca2+ release from intracellular InsP3 sensitive stores can be modulated in a differential manner. Namely, discrete elevations of cytosolic Ca2+ switch on the Ca2+ release whereas higher Ca2+ concentrations dampen the release. These results suggest a functional coupling between P-type VDCCs and InsP3 receptors.

Regulation by protein kinase-C of putative P-type Ca channels expressed in Xenopus oocytes from cerebellar mRNA.

Xenopus oocytes injected with rat cerebellar mRNA expressed functional voltage-dependent Ca channels detected as an inward Ba current (IBa). The pharmacological resistance to dihydropyridines and omega-conotoxin together with the blockade obtained with Agelenopsis aperta venom suggest that these channels could be somehow assimilated to P-type Ca channels. The precise nature of the transplanted Ca channels was assessed by hybrid-arrest experiments using a specific oligonucleotide antisense-derivated from the recently cloned alpha 1-subunit of P channels (BI-1 clone). In addition, we demonstrate that exogenous Ca channel activity was enhanced by two different PKC activators (a phorbol ester and a structural analog to diacylglycerol). The general electrophysiological and pharmacological properties of the stimulated Ca channels remain unchanged. This potentiation induced by PKC activators is antagonized by a PKC inhibitor (staurosporine) and by a monoclonal antibody directed against PKC. It is concluded that P-type Ca channels are potentially regulated by PKC phosphorylation and the functional relevance of this intracellular pathway is discussed.