Dual phosphorylations underlie modulation of unitary KCNQ K+ channels by Src tyrosine kinase

Yang Li, Paul Langlais, Nikita Gamper, Feng Liu, Mark S Shapiro

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)45399-45407
Number of pages9
JournalJournal of Biological Chemistry
Volume279
Issue number44
DOIs
StatePublished - Oct 29 2004

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Phosphorylation
src-Family Kinases
Modulation
Tyrosine
Assays
Mutagenesis
Biochemistry
Clamping devices
Cell membranes
G-Protein-Coupled Receptors
Biotinylation
Ion Channels
Immunoprecipitation
Membranes
Kinetics
Cell Membrane
Mutation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Dual phosphorylations underlie modulation of unitary KCNQ K+ channels by Src tyrosine kinase. / Li, Yang; Langlais, Paul; Gamper, Nikita; Liu, Feng; Shapiro, Mark S.

In: Journal of Biological Chemistry, Vol. 279, No. 44, 29.10.2004, p. 45399-45407.

Research output: Contribution to journalArticle

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