title: Modulation of human cardiac transient outward potassium current by EGFR tyrosine kinase and Src-family kinases.
abstract: OBJECTIVE
  The human cardiac transient outward K(+) current I(to) (encoded by Kv4.3 or KCND3) plays an important role in phase 1 rapid repolarization of cardiac action potentials in the heart. However, modulation of I(to) by intracellular signal transduction is not fully understood. The present study was therefore designed to determine whether/how human atrial I(to) and hKv4.3 channels stably expressed in HEK 293 cells are regulated by protein tyrosine kinases (PTKs).
  
  
  RESULTS
  Whole-cell patch voltage-clamp, immunoprecipitation, western blotting, and site-directed mutagenesis approaches were employed in the present study. We found that human atrial I(to) was inhibited by the broad-spectrum PTK inhibitor genistein, the selective epidermal growth factor receptor (EGFR) kinase inhibitor AG556, and the Src-family kinases inhibitor PP2. The inhibitory effect was countered by the protein tyrosine phosphatase inhibitor orthovanadate. In HEK 293 cells stably expressing human KCND3, genistein, AG556, and PP2 significantly reduced the hKv4.3 current, and the reduction was antagonized by orthovanadate. Interestingly, orthovanadate also reversed the reduced tyrosine phosphorylation level of hKv4.3 channels by genistein, AG556, or PP2. Mutagenesis revealed that the hKv4.3 mutant Y136F lost the inhibitory response to AG556, while Y108F lost response to PP2. The double-mutant Y108F-Y136F hKv4.3 channels showed no response to either AG556 or PP2.
  
  
  CONCLUSIONS
  Our results demonstrate that human atrial I(to) and cloned hKv4.3 channels are modulated by EGFR kinase via phosphorylation of the Y136 residue and by Src-family kinases via phosphorylation of the Y108 residue; tyrosine phosphorylation of the channel may be involved in regulating cardiac electrophysiology.
authors.affiliation: Department of Medicine, Li Ka Shing Faculty of Medicine,  The University of Hong Kong, L4-59, Laboratory Block, FMB, 21 Sassoon Road, Pokfulam, Hong Kong,  China.
authors.forename: Yan-Hui
authors.initials: YH
authors.lastname: Zhang
authors.forename: Wei
authors.initials: W
authors.lastname: Wu
authors.forename: Hai-Ying
authors.initials: HY
authors.lastname: Sun
authors.forename: Xiu-Ling
authors.initials: XL
authors.lastname: Deng
authors.forename: Lik-Cheung
authors.initials: LC
authors.lastname: Cheng
authors.forename: Xin
authors.initials: X
authors.lastname: Li
authors.forename: Hung-Fat
authors.initials: HF
authors.lastname: Tse
authors.forename: Chu-Pak
authors.initials: CP
authors.lastname: Lau
authors.forename: Gui-Rong
authors.initials: GR
authors.lastname: Li
mesh_terms: D000200:Action Potentials / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D066246:ErbB Receptors / Q000378:metabolism*; D019833:Genistein / Q000494:pharmacology; D057809:HEK293 Cells; D006325:Heart Atria / Q000166:cytology; D006801:Humans; D016297:Mutagenesis, Site-Directed; D032383:Myocytes, Cardiac / Q000166:cytology / Q000502:physiology*; D018408:Patch-Clamp Techniques; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051672:Shal Potassium Channels / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology; D014443:Tyrosine / Q000378:metabolism; D020032:Tyrphostins / Q000494:pharmacology; D019061:src-Family Kinases / Q000378:metabolism*
chemical_list: C497021:KCND3 protein, human; D047428:Protein Kinase Inhibitors; D051672:Shal Potassium Channels; D020032:Tyrphostins; C098981:AG 556; D014443:Tyrosine; D019833:Genistein; C512478:EGFR protein, human; D066246:ErbB Receptors; D019061:src-Family Kinases