ATP-dependent activation of the intermediate conductance, Ca2+-activated K+ channel, hIK1, is conferred by a C-terminal domain (vol 276, pg 10963, 2001)

We previously demonstrated that hIK1 is activated directly by ATP in excise d, inside-out patches in a protein kinase A inhibitor 5-24 dependent manner , suggesting a role for phosphorylation in the regulation of this Ca2+-depe ndent channel. However, mutation of the single consensus cAMP-dependent pro tein kinase phosphorylation site (S334A) failed to modify the response of h IK1 to ATP (Gerlach, A. C., Gangopadhyay, N. N., and Devor, D. C. (2000) J. Biol Chem. 275, 585-598). Here we demonstrate that ATP does not similarly activate the highly homologous Ca2+-dependent K+ channels, hSK1, rSK2, and rSK3, To define the region of hIK1 responsible for the ATP-dependent regula tion, we generated a series of hIK1 truncations and hIK1/rSK2 chimeras. ATP did not activate a chimera containing the N terminus plus S1-S4 from hIK1. In contrast, ATP activated a chimera containing the hIK1 C-terminal amino acids His(299)- Lys(427). Furthermore, truncation of hIK1 at Leu(414) resul ted in an ATP-dependent channel, whereas larger truncations of hIK1 failed to express. Additional hIK1/rSK2 chimeras defined the minimal region of hIK 1 required to confer complete ATP sensitivity as including amino acids Arg( 355)-Ala(413). An alanine scan of all nonconserved serines and threonines w ithin this region failed to alter the response of hIK1 to ATP, suggesting t hat hIK1 itself is not directly phosphorylated. Additionally, substitution of amino acids Arg(355)-Met(368) Of hIK1 into the corresponding region of r SK2 resulted in an ATP-dependent activation, which was similar to 50% of th at of hIK1, These results demonstrate that amino acids Arg(355)-Ala(413) wi thin the C terminus of hIK1 confer sensitivity to ATP. Finally, we demonstr ate that the ATP-dependent phosphorylation of hIK1 or an associated protein is independent of Ca2+.