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Protein kinase C isoform antagonism controls BNaC2 (ASIC1) function
Bakhrom K Berdiev1, Jiazeng Xia, Biljana Jovov
1Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
Abstract:
We explored the involvement of protein kinase C (PKC) and its isoforms in the regulation of BNaC2. Reverse transcriptase PCR evaluation of PKC isoform expression at the level of mRNA revealed the presence of alpha and epsilon/epsilon' in all glioma cell lines analyzed; most, but not all cell lines expressed delta and zeta. No messages were found for the betaI and betaII isotypes of PKC in the tumor cells. Normal astrocytes expressed beta but not gamma. The essential features of these results were confirmed at the protein level by Western analysis. This disproportionate pattern of PKC isoform expression in glioma cell lines was further echoed in the functional effects of these PKC isoforms on BNaC2 activity in bilayers. PKC holoenzyme or the combination of PKCbetaI and PKCbetaII isoforms inhibited BNaC2. Neither PKCepsilon nor PKCzeta or their combination had any effect on BNaC2 activity in bilayers. The inhibitory effect of the PKCbetaI and PKCbetaII mixture on BNaC2 activity was abolished by a 5-fold excess of a PKCepsilon and PKCzeta combination. PKC holoenzymes, PKCbetaI, PKCbetaII, PKCdelta, PKCepsilon, and PKCzeta phosphorylated BNaC2 in vitro. In patch clamp experiments, the combination of PKCbetaI and PKCbetaII inhibited the basally activated inward Na(+) conductance. The variable expression of the PKC isotypes and their functional antagonism in regulating BNaC2 activity support the idea that the participation of multiple PKC isotypes contributes to the overall activity of BNaC2.
Insights
Protein kinase C (PKC) isoforms differentially regulate BNaC2 channels. Specific PKC isoforms inhibit BNaC2, while others have no effect, suggesting complex channel modulation in glioma cells.
Area of Science:
- Neuroscience
- Molecular Biology
- Cell Signaling
Background:
- Protein kinase C (PKC) plays a role in cellular signaling pathways.
- BNaC2 channels are involved in regulating ion conductance.
Purpose of the Study:
- To investigate the role and expression of various protein kinase C (PKC) isoforms in the regulation of BNaC2 channels.
- To understand the functional impact of different PKC isoforms on BNaC2 activity in glioma cells.
Main Methods:
- Reverse transcriptase PCR (RT-PCR) for mRNA expression analysis of PKC isoforms.
- Western blot analysis for protein expression confirmation.
- Bilayer and patch-clamp electrophysiology to assess BNaC2 channel activity.
- In vitro phosphorylation assays.
Main Results:
- Glioma cells predominantly express PKC alpha, epsilon/epsilon', delta, and zeta isoforms at mRNA and protein levels, with limited expression of betaI/betaII.
- PKC holoenzyme and PKCbetaI/betaII isoforms inhibited BNaC2 activity, while PKCepsilon and PKCzeta had no effect.
- PKCbetaI/betaII inhibition of BNaC2 was counteracted by PKCepsilon/PKCzeta.
- PKC isoforms phosphorylated BNaC2 in vitro, and PKCbetaI/betaII inhibited basal Na+ conductance.
Conclusions:
- Differential expression and functional antagonism of PKC isoforms regulate BNaC2 activity.
- Multiple PKC isoforms contribute to the complex modulation of BNaC2 channel function in glioma cells.