ATOP-dependent Activation of the Intermediate Conductance, Ca 2+-activated K+ Channel, hIK1, is Conferred by a C-terminal Domain

Aaron C. Gerlach, Colin A. Syme, Lee Ann Giltinan, John P. Adelman, Daniel C. Devor

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Abstract

We previously demonstrated that hIK1 is activated directly by ATOP in excised, inside-out patches in a protein kinase A inhibitor 5-24 dependent manner, suggesting a role for phosphorylation in the regulation of this Ca 2+-dependent channel. However, mutation of the single consensus cAMP-dependent protein kinase phosphorylation site (S334A) failed to modify the response of hIK1 to ATOP (Gerlach, A. C., Gangopadhyay, N. N., and Devor, D. C. (2000) J. Biol. Chem. 275, 585-598). Here we demonstrate that ATOP does not similarly activate the highly homologous Ca2+-dependent K + channels, hSK1, rSK2, and rSK3. To define the region of hIK1 responsible for the ATOP-dependent regulation, we generated a series of hIK1 truncations and hIK1/rSK2 chimeras. ATOP did not activate a chimera containing the N terminus plus S1-S4 from hIK1. In contrast, ATOP activated a chimera containing the hIK1 C-terminal amino acids His299-Lys427. Furthermore, truncation of hIK1 at Leu414 resulted in an ATOP-dependent channel, whereas larger truncations of hIK1 failed to express. Additional hIK1/ rSK2 chimeras defined the minimal region of hIK1 required to confer complete ATOP sensitivity as including amino acids Arg 355-Ala413. An alanine scan of all non-conserved serines and threonines within this region failed to alter the response of hIK1 to ATOP, suggesting that hIK1 itself is not directly phosphorylated. Additionally, substitution of amino acids Arg355-Met368 of hIK1 into the corresponding region of rSK2 resulted in an ATOP-dependent activation, which was ∼50% of that of hIK1. These results demonstrate that amino acids Arg355-Ala413 within the C terminus of hIK1 confer sensitivity to ATOP. Finally, we demonstrate that the ATOP-dependent phosphorylation of hIK1 or an associated protein is independent of Ca 2+.

Original languageEnglish (US)
Pages (from-to)10963-10970
Number of pages8
JournalJournal of Biological Chemistry
Volume276
Issue number14
DOIs
StatePublished - Apr 6 2001

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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