Abstract
The human ether-a-go-go - related gene (hERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel (IKr), which is important for cardiac repolarization. Reduction of hERG current due to genetic mutations or drug interferences causes long QT syndrome (LQTS), leading to cardiac arrhythmias and sudden death. To date, there is no effective therapeutic method to restore or enhance hERG channel function. Using cell biology and electrophysiological methods, we found that muscarinic receptor agonist carbachol increased the expression and function of hERG, but not EAG or Kv1.5 channels stably expressed in HEK cells. The carbachol-mediated increase in hERG expression was abolished by the selective M3 antagonist 4-DAMP but not by the M2 antagonist AF-DX 166. Treatment of cells with carbachol reduced the hERG-ubiquitin interaction and slowed the rate of hERG degradation. We previously showed that the E3 ubiquitin ligase Nedd4-2 mediates degradation of hERG channels. Here, we found that disrupting the Nedd4-2 binding domain in hERG completely eliminated the effect of carbachol on hERG channels. Carbachol treatment enhanced the phosphorylation level, but not the total level, of Nedd4-2. Blockade of the PKC pathway abolished the carbachol-induced enhancement of hERG channels. We conclude that muscarinic activation increases hERG channel expression by phosphorylating Nedd4-2 via the PKC pathway.
- Muscarinic cholinergic
- Ion channel regulation
- Potassium channels
- Protein Kinase C
- Receptor degradation
- Ubiquitination
- Regulation of gene expression
- Patch clamp methods
- The American Society for Pharmacology and Experimental Therapeutics