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Receptor-coupled regulation of K+ channel N-type inactivation
I Velasco1, E J Beck, M Covarrubias
1Department of Pathology, Anatomy and Cell Biology, Jefferson Medical College, Philadelphia, PA 19107, USA.
Abstract:
Phosphorylation of the inactivation gate of a K+ channel (Kv3.4) by protein kinase C (PKC) slows rapid N-type inactivation. To demonstrate that such an effect could occur under more physiological conditions, Kv3.4 and a metabotropic serotonin (5-HT) receptor were coexpressed in Xenopus oocytes. Application of 5-HT 10 microM to these oocytes produced two main effects: 1) Enhanced activity of endogenous Ca(++)-dependent Cl- channels; and 2) Kv3.4 currents exhibited significantly slower inactivation than the control currents (time constants at +50 mV: 7.1 +/- 0.6 ms and 14.7 +/- 3 ms, before and after 5-HT, respectively). These results are consistent with the presence of receptor-coupled activation of phospholipase C. 5-HT had little or no effect on Kv3.4 current kinetics when four N-terminal serines were mutated to alanine. Peak currents exhibited, however, a slow run-down. This study demonstrates that physiological activation of PKC may regulate K+ channel inactivation by a direct action.
Insights
Protein kinase C (PKC) phosphorylation of Kv3.4 potassium channels slows inactivation. This study shows serotonin receptor activation in Xenopus oocytes mimics this effect, demonstrating physiological regulation of K+ channel inactivation.
Area of Science:
- Neuroscience
- Molecular Biology
- Ion Channel Physiology
Background:
- Protein kinase C (PKC) is known to phosphorylate the Kv3.4 potassium channel, affecting its inactivation.
- Understanding the physiological relevance of this phosphorylation requires studying it under conditions that mimic cellular signaling pathways.
Purpose of the Study:
- To investigate if activation of a metabotropic serotonin (5-HT) receptor can trigger PKC-mediated regulation of Kv3.4 channel inactivation in a physiological context.
- To determine the role of specific serine residues in the N-terminus of Kv3.4 channels in 5-HT-induced modulation.
Main Methods:
- Coexpression of Kv3.4 channels and a 5-HT receptor in Xenopus oocytes.
- Electrophysiological recordings of Kv3.4 currents before and after 5-HT application.
- Site-directed mutagenesis of N-terminal serine residues in Kv3.4 channels.
Main Results:
- Application of 10 microM 5-HT to oocytes expressing Kv3.4 and the 5-HT receptor significantly slowed Kv3.4 current inactivation.
- 5-HT also enhanced endogenous Ca(++)-dependent Cl- channel activity, suggesting phospholipase C activation.
- Mutation of four N-terminal serines to alanine abolished the effect of 5-HT on Kv3.4 inactivation kinetics, although peak currents showed run-down.
Conclusions:
- Physiological activation of PKC, triggered by 5-HT receptor stimulation, directly regulates K+ channel inactivation.
- The N-terminal serine residues of Kv3.4 channels are critical for this PKC-mediated modulation.
- This finding provides insight into how G protein-coupled receptor signaling can influence neuronal excitability through modulation of potassium channel function.