Regulation by protein kinase-C of putative P-type Ca channels expressed in Xenopus oocytes from cerebellar mRNA
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F Fournier1, P Charnet, E Bourinet
1Laboratoire de Neurobiologie Cellulaire, Université de Picardie, Faculté des Sciences, Amiens, France.
Abstract:
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.
Insights
Rat cerebellar mRNA in Xenopus oocytes produced functional P-type calcium channels. Protein kinase C (PKC) activators enhanced channel activity, suggesting PKC phosphorylation regulates these calcium channels.
Area of Science:
- Neuroscience
- Molecular Biology
- Biophysics
Background:
- Voltage-dependent calcium channels are crucial for neuronal function.
- Specific subtypes of calcium channels exhibit distinct pharmacological and molecular properties.
Purpose of the Study:
- To characterize the type of voltage-dependent calcium channels expressed from rat cerebellar mRNA in Xenopus oocytes.
- To investigate the potential regulation of these calcium channels by protein kinase C (PKC).
Main Methods:
- Xenopus oocyte expression system.
- Electrophysiological recordings to detect barium currents (IBa).
- Pharmacological profiling using dihydropyridines, omega-conotoxin, and Agelenopsis aperta venom.
- Hybrid-arrest experiments with antisense oligonucleotides targeting the alpha 1-subunit of P-type calcium channels.
- Treatment with PKC activators (phorbol ester, diacylglycerol analog) and inhibitors (staurosporine, anti-PKC antibody).
Main Results:
- Expression of functional voltage-dependent calcium channels in Xenopus oocytes.
- Pharmacological properties suggested assimilation to P-type calcium channels.
- Hybrid-arrest experiments confirmed the identity as P-type calcium channels.
- PKC activators significantly enhanced exogenous calcium channel activity without altering channel properties.
- PKC inhibitor and antibody antagonized the potentiation effect.
Conclusions:
- Rat cerebellar mRNA encodes P-type calcium channels expressed in Xenopus oocytes.
- P-type calcium channels are subject to regulation by PKC phosphorylation.
- PKC-mediated phosphorylation represents a potential intracellular pathway for modulating P-type calcium channel function.