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Protein kinase C inhibits Kv1.1 potassium channel function
1Department of Physiology and Program in Neuroscience, University of Minnesota, Minneapolis, Minnesota 55455, USA. bolan007@tc.umn.edu
Abstract:
The regulation by protein kinase C (PKC) of recombinant voltage-gated potassium (K) channels in frog oocytes was studied. Phorbol 12-myristate 13-acetate (PMA; 500 nM), an activator of PKC, caused persistent and large (up to 90%) inhibition of mouse, rat, and fly Shaker K currents. K current inhibition by PMA was blocked by inhibitors of PKC, and inhibition was not observed in control experiments with PMA analogs that do not activate PKC. However, site-directed substitution of potential PKC phosphorylation sites in the Kv1.1 protein did not prevent current inhibition by PMA. Kv1.1 current inhibition was also not accompanied by changes in macroscopic activation kinetics or in the conductance-voltage relationship. In Western blots, Kv1.1 membrane protein was not significantly reduced by PKC activation. The injection of oocytes with botulinum toxin C3 exoenzyme blocked the PMA inhibition of Kv1. 1 currents. These data are consistent with the hypothesis that PKC-mediated inhibition of Kv1.1 channel function occurs by a novel mechanism that requires a C3 exoenzyme substrate but does not alter channel activation gating or promote internalization of the channel protein.
Insights
Protein kinase C (PKC) activation inhibits potassium (K) channel function via a novel mechanism. This inhibition requires a C3 exoenzyme substrate but does not affect channel gating or protein levels.
Area of Science:
- Molecular biology
- Neuroscience
- Ion channel physiology
Background:
- Protein kinase C (PKC) is a key regulator of cellular processes.
- Voltage-gated potassium (K) channels play crucial roles in neuronal excitability.
- The precise mechanisms of PKC-mediated K channel regulation are not fully understood.
Purpose of the Study:
- To investigate the mechanism by which PKC regulates recombinant Shaker K channels (Kv1.1).
- To determine if PKC phosphorylation sites are involved in the observed inhibition.
- To explore the role of C3 exoenzyme substrates in PKC-mediated channel modulation.
Main Methods:
- Heterologous expression of Kv1.1 channels in Xenopus oocytes.
- Electrophysiological recordings (whole-cell patch clamp) to measure K currents.
- Pharmacological manipulation using PKC activators (PMA) and inhibitors.
- Site-directed mutagenesis of potential PKC phosphorylation sites.
- Western blot analysis to assess protein levels.
- Functional assays involving botulinum toxin C3 exoenzyme.
Main Results:
- Phorbol 12-myristate 13-acetate (PMA), a PKC activator, caused significant inhibition (up to 90%) of Kv1.1 currents.
- Inhibition was PKC-dependent, as blocked by PKC inhibitors and absent with inactive PMA analogs.
- Mutating putative PKC phosphorylation sites did not prevent PMA-induced inhibition.
- Kv1.1 current inhibition was not associated with changes in activation kinetics or voltage-dependence.
- Kv1.1 protein levels at the membrane were not significantly reduced by PKC activation.
- Botulinum toxin C3 exoenzyme injection blocked PMA-induced Kv1.1 current inhibition.
Conclusions:
- PKC-mediated inhibition of Kv1.1 channels occurs through a novel pathway.
- This mechanism requires a substrate for C3 exoenzyme.
- The inhibition does not involve alterations in channel gating properties or protein degradation/internalization.