Identification and Functional Characterization of Protein Kinase A-catalyzed Phosphorylation of Potassium Channel Kv1.2 at Serine 449

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dc.contributor.authorJohnson, Rosalyn P.
dc.contributor.authorEl-Yazbi, Ahmed F.
dc.contributor.authorHughes, Morgan F.
dc.contributor.authorSchriemer, David C.
dc.contributor.authorWalsh, Emma J.
dc.contributor.authorWalsh, Michael P.
dc.contributor.authorCole, William C.
dc.date.accessioned2017-08-17T21:10:10Z
dc.date.available2017-08-17T21:10:10Z
dc.date.issued2009-04-22
dc.description.abstractVascular 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.en_US
dc.description.grantingagencyCanadian Institutes of Health Research Granten_US
dc.description.refereedYesen_US
dc.identifier.citationJohnson, R. P., El-Yazbi, A. F., Hughes, M. F., Schriemer, D. C., Walsh, E. J., Walsh, M. P., & Cole, W. C. (2009). Identification and functional characterization of protein kinase A-catalyzed phosphorylation of potassium channel Kv1.2 at serine 449. Journal of Biological Chemistry, 284(24), 16562-16574. doi:10.1074/jbc.M109.010918en_US
dc.identifier.doi10.1074/jbc.M109.010918
dc.identifier.doihttp://dx.doi.org/10.11575/PRISM/33798
dc.identifier.grantnumberMOP-10569en_US
dc.identifier.urihttp://hdl.handle.net/1880/52194
dc.language.isoenen_US
dc.publisherJournal of Biological Chemistryen_US
dc.publisher.institutionUniversity of Calgaryen_US
dc.publisher.urlhttp://www.jbc.org/en_US
dc.subjectPhosphorylation - physiologyen_US
dc.subjectCyclic AMP-Dependent Protein Kinases - metabolismen_US
dc.subjectKv1.2 Potassium Channel - geneticsen_US
dc.subjectKidney - cytologyen_US
dc.subjectKv1.2 Potassium Channel - chemistryen_US
dc.subjectSerine - metabolismen_US
dc.titleIdentification and Functional Characterization of Protein Kinase A-catalyzed Phosphorylation of Potassium Channel Kv1.2 at Serine 449en_US
dc.typejournal article
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