Mass spectrometric analysis reveals a functionally important PKA phosphorylation site in a Kir3 channel subunit

Radda Rusinova, Yu Ming Albert Shen, Georgia Dolios, Julio Padovan, Heyi Yang, Madeleine Kirchberger, Rong Wang, Diomedes E. Logothetis

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13 Scopus citations


Phosphorylation of the Kir3 channel by cAMP-dependent protein kinase (PKA) potentiates activity and strengthens channel-PIP2 interactions, whereas phosphorylation by protein kinase C (PKC) exerts the opposite effects (Keselman et al., Channels 1:113-123, 2007; Lopes et al., Channels 1:124-134, 2007). Unequivocal identification of phosphorylated residues in ion channel proteins has been difficult, but recent advances in mass spectrometry techniques have allowed precise identification of phosphorylation sites (Park et al., Science 313:976-979, 2006). In this study, we utilized mass spectrometry to identify phosphorylation sites within the Kir3.1 channel subunit. We focused on the Kir3.1 C-terminal cytosolic domain that has been reported to be regulated by several modulators. In vitro phosphorylation by PKA exhibited a convincing signal upon treatment with a phosphoprotein stain. The phosphorylated C terminus was subjected to mass spectrometric analysis using matrix-assisted lased desorption/ionization-time of flight mass spectroscopy (MS). Peptides whose mass underwent a shift corresponding to addition of a phosphate group were then subjected to tandem MS (MS/MS) in order to confirm the modification and determine its precise location. Using this approach, we identified S385 as an in vitro phosphorylation site. Mutation of this residue to alanine resulted in a reduced sensitivity of Kir3.1*currents to H89 and Forskolin, confirming an in vivo role for this novel site of the Kir3.1 channel subunit in its regulation by PKA.

Original languageEnglish
Pages (from-to)303-314
Number of pages12
JournalPflugers Archiv European Journal of Physiology
Issue number2
StatePublished - Jun 2009


  • G-protein-gated inwardly rectifying potassium channels
  • Ion channel regulation
  • Mass spectrometry
  • Phosphorylation
  • Posttranslational modification
  • Protein kinase A
  • Tandem MS


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