Dual phosphorylations underlie modulation of unitary KCNQ K(+) channels by Src tyrosine kinase.
Src tyrosine kinase suppresses KCNQ (M-type) K(+) channels in a subunit-specific manner representing a mode of modulation distinct from that involving G protein-coupled receptors. We probed the molecular and biophysical mechanisms of this modulation using mutagenesis, biochemistry, and both whole-cell and single channel modes of patch clamp recording. Immunoprecipitation assays showed that Src associates with KCNQ2-5 subunits but phosphorylates only KCNQ3-5. Using KCNQ3 as a background, we found that mutation of a tyrosine in the amino terminus (Tyr-67) or one in the carboxyl terminus (Tyr-349) abolished Src-dependent modulation of heterologously expressed KCNQ2/3 heteromultimers. The tyrosine phosphorylation was much weaker for either the KCNQ3-Y67F or KCNQ3-Y349F mutants and wholly absent in the KCNQ3-Y67F/Y349F double mutant. Biotinylation assays showed that Src activity does not alter the membrane abundance of channels in the plasma membrane. In recordings from cell-attached patches containing a single KCNQ2/3 channel, we found that Src inhibits the open probability of the channels. Kinetic analysis was consistent with the channels having two discrete open times and three closed times. Src activity reduced the durations of the longest open time and lengthened the longest closed time of the channels. The implications for the mechanisms of channel regulation by the dual phosphorylations on both channel termini are discussed.
Department of Physiology, University of Texas Health Science Center, San Antonio, Texas 78229, USA.
Yang
Y
Li
Paul
P
Langlais
Nikita
N
Gamper
Feng
F
Liu
Mark S
MS
Shapiro
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D006801:Humans; D015640:Ion Channel Gating; D051656:KCNQ Potassium Channels; D051658:KCNQ2 Potassium Channel; D051659:KCNQ3 Potassium Channel; D010766:Phosphorylation; D024642:Potassium Channels, Voltage-Gated / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D019061:src-Family Kinases / Q000502:physiology*
D051656:KCNQ Potassium Channels; D051658:KCNQ2 Potassium Channel; C496914:KCNQ2 protein, human; D051659:KCNQ3 Potassium Channel; C496916:KCNQ3 protein, human; C117367:KCNQ4 protein, human; C412231:KCNQ5 protein, human; C496915:Kcnq2 protein, rat; C496918:Kcnq3 protein, rat; C501028:Kcnq5 protein, rat; D024642:Potassium Channels, Voltage-Gated; D019061:src-Family Kinases