Subunit-specific modulation of KCNQ potassium channels by Src tyrosine kinase.
We studied regulation by c-Src tyrosine kinase (Src) of KCNQ1-5 channels heterologously expressed in Chinese hamster ovary (CHO) cells and of native M current in rat sympathetic neurons. Using whole-cell patch clamp, we found that Src modulates currents from KCNQ3, KCNQ4, and KCNQ5 homomultimers, KCNQ2/3 heteromultimers and native M current, but not currents from KCNQ1 or KCNQ2 homomultimers. Src overexpression had two effects: a decrease of current amplitude (4- to 15-fold for cloned channels and approximately 3-fold for M current) and a slowing of activation kinetics by 2-fold. Both Src actions were mostly reversed by bath application of the Src inhibitors erbstatin (20 microm) and PP2 (200 nm), and mimicked by the tyrosine phosphatase inhibitor sodium vanadate (100 microm). Immunoprecipitation and immunoblot analysis showed Src-dependent phosphotyrosine signals associated with KCNQ3, KCNQ4, and KCNQ5 but not with KCNQ1 or KCNQ2 that may be tyrosine phosphorylation of the channel subunits. Expression of a dominant negative Src that cannot phosphorylate substrates had no effect on the current and did not induce phosphotyrosine signals associated with KCNQ3-5 subunits, further indicating that Src actions on KCNQ currents are mediated by tyrosine phosphorylation. Immunostaining and confocal analysis showed no effect of Src overexpression on the abundance of KCNQ3 protein in CHO cells. Finally, experiments using cloned KCNQ2/3 channels, Src and M(1) muscarinic receptors, and sympathetic neurons demonstrated that the actions on KCNQ channels by Src and by muscarinic agonists use distinct mechanisms.
Gamper
Nikita
N
Department of Physiology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229, USA.
Stockand
James D
JD
Shapiro
Mark S
MS
D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D004553:Electric Conductivity; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051658:KCNQ2 Potassium Channel; D051659:KCNQ3 Potassium Channel; D007700:Kinetics; D018721:Muscarinic Agonists / Q000494:pharmacology; D009154:Mutation; D009474:Neurons / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D015221:Potassium Channels / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D024642:Potassium Channels, Voltage-Gated; D021122:Protein Subunits; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017783:Superior Cervical Ganglion / Q000166:cytology / Q000502:physiology; D014443:Tyrosine / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology
D004791:Enzyme Inhibitors; D051658:KCNQ2 Potassium Channel; D051659:KCNQ3 Potassium Channel; C496915:Kcnq2 protein, rat; C496918:Kcnq3 protein, rat; D018721:Muscarinic Agonists; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D021122:Protein Subunits; D014638:Vanadates; D014443:Tyrosine; D016392:Proto-Oncogene Proteins pp60(c-src)