Regulation of human ether-a-go-go-related gene potassium channels by EGFR kinase and Src-related tyrosine

Abstract

Introduction: Protein tyrosine kinases (PTKs) transduce extracellular signals mediate events such as proliferation, cytoskeletal rearrangement, and coordination of physiological responses, and also regulate ion channels. The present study was designed to investigate whether PTKs regulate human ether-a`-go-go-related gene (hERG) channels. Methods: Whole-cell patch clamp technique was applied to record hERG channel current (IhERG) in HEK 293 cell line stably expressing cloned and mutated hERG channels. Immunoprecipitation and Western blot analysis were used to determine tyrosine phosphorylation level of hERG channels. Results: The broad-spectrum PTK inhibitor genistein (30 lM), the highly selective EGFR (epidermal growth factor receptor) kinase inhibitor AG556 (10 lM) and the Src-family kinase inhibitor PP2 (10 lM) remarkably inhibited IhERG, and the effects were significantly reversed by the protein tyrosine phosphatase (PTP) inhibitor orthovanadate (1 mM). Immunoprecipitation and western blot analysis demonstrated that tyrosine phosphorylation level of hERG channels was reduced by genistein, AG556, and PP2, and the reduction of the phosphorylation level of hERG channels by genistein, PP2 was significantly antagonized by orthovanadate. Single point mutation(s) of Y475A and/or Y611A dramatically reduced the inhibitory effect of IhERG by PP2 and AG556. Conclusions: Our results demonstrate the novel information that IhERG is modulated not only by Src-family kinases, but also by ERFR kinases. Y475 and/or Y611 are likely preferred phosphorylation sites.