title: Identification and functional characterization of protein kinase A-catalyzed phosphorylation of potassium channel Kv1.2 at serine 449.
abstract: Vascular smooth muscle Kv1 delayed rectifier K+ channels (KDR) containing Kv1.2 control membrane potential and thereby regulate contractility. Vasodilatory agonists acting via protein kinase A (PKA) enhance vascule smooth muscle Kv1 activity, but the molecular basis of this regulation is uncertain. We characterized the role of a C-terminal phosphorylation site, Ser-449, in Kv1.2 expressed in HEK 293 cells by biochemical and electrophysiological methods. We found that 1) in vitro phosphorylation of Kv1.2 occurred exclusively at serine residues, 2) one major phosphopeptide that co-migrated with 449pSASTISK was generated by proteolysis of in vitro phosphorylated Kv1.2, 3) the peptide 445KKSRSASTISK exhibited stoichiometric phosphorylation by PKA in vitro, 4) matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectroscopy (MS) and MS/MS confirmed in vitro Ser-449 phosphorylation by PKA, 5) in situ phosphorylation at Ser-449 was detected in HEK 293 cells by MALDI-TOF MS followed by MS/MS. MIDAS (multiple reaction monitoring-initiated detection and sequencing) analysis revealed additional phosphorylated residues, Ser-440 and Ser-441, 6) in vitro 32P incorporation was significantly reduced in Kv1.2-S449A, Kv1.2-S449D, and Kv1.2-S440A/S441A/S449A mutant channels, but Kv1.2-S440A/S441A was identical to wild-type Kv1.2 (Kv1.2-WT), and 7) bath applied 8-Br-cAMP or dialysis with PKA catalytic subunit (cPKA) increased Kv1.2-WT but not Kv1.2-S449A current amplitude. cPKA increased Kv1.2-WT current in inside-out patches. Rp-CPT-cAMPS reduced Kv1.2-WT current, blocked the increase due to 8-Br-cAMP, but had no effect on Kv1.2-S449A. cPKA increased current due to double mutant Kv1.2-S440A/S441A but had no effect on Kv1.2-S449D or Kv1.2-S440A/S441A/S449A. We conclude that Ser-449 in Kv1.2 is a site of PKA phosphorylation and a potential molecular mechanism for Kv1-containing KDR channel modulation by agonists via PKA activation.
authors.affiliation: From the Smooth Muscle Research Group, Calgary, Alberta T2N 4N1, Canada.
authors.forename: Rosalyn P
authors.initials: RP
authors.lastname: Johnson
authors.affiliation: From the Smooth Muscle Research Group, Calgary, Alberta T2N 4N1, Canada.
authors.forename: Ahmed F
authors.initials: AF
authors.lastname: El-Yazbi
authors.affiliation: Southern Alberta Mass Spectrometry Centre, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
authors.forename: Morgan F
authors.initials: MF
authors.lastname: Hughes
authors.affiliation: Southern Alberta Mass Spectrometry Centre, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
authors.forename: David C
authors.initials: DC
authors.lastname: Schriemer
authors.affiliation: From the Smooth Muscle Research Group, Calgary, Alberta T2N 4N1, Canada.
authors.forename: Emma J
authors.initials: EJ
authors.lastname: Walsh
authors.affiliation: From the Smooth Muscle Research Group, Calgary, Alberta T2N 4N1, Canada.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Walsh
authors.affiliation: From the Smooth Muscle Research Group, Calgary, Alberta T2N 4N1, Canada. Electronic address: wcole@ucalgary.ca.
authors.forename: William C
authors.initials: WC
authors.lastname: Cole
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000166:cytology; D051663:Kv1.2 Potassium Channel / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008564:Membrane Potentials / Q000502:physiology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D010766:Phosphorylation / Q000502:physiology; D017434:Protein Structure, Tertiary; D011817:Rabbits; D012694:Serine / Q000378:metabolism
chemical_list: D051663:Kv1.2 Potassium Channel; D012694:Serine; D017868:Cyclic AMP-Dependent Protein Kinases