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J. Biol. Chem., Vol. 278, Issue 19, 16690-16697, May 9, 2003
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From the We previously demonstrated that the
endogenously expressed human intermediate conductance,
Ca2+-activated K+ channel (hIK1) was
inhibited by arachidonic acid (AA) (Devor, D. C., and Frizzell,
R. A. (1998) Am. J. Physiol. 274, C138-C148). Here we demonstrate, using the excised, inside-out patch-clamp technique, that hIK1, heterologously expressed in HEK293 cells, is
inhibited 82 ± 2% (n = 16) with 3 µM AA, being half-maximally inhibited (IC50)
at 1.4 ± 0.7 µM. In contrast, AA does not inhibit the Ca2+-dependent, small conductance
K+ channel, rSK2, another member of the KCNN
gene family. Therefore, we utilized chimeric hIK1/rSK2 channels to
define the AA binding domain on hIK1 to the S5-Pore-S6 region of the
channel. Subsequent site-directed mutagenesis revealed that mutation of
Thr250 to Ser (T250S) resulted in a channel with limited
sensitivity to block by AA (8 ± 2%, n = 8),
demonstrating that Thr250 is a key molecular determinant
for the inhibition of hIK1 by AA. Likewise, when Val275 in
S6 was mutated to Ala (V275A) AA inhibited only 43 ± 11%
(n = 9) of current flow. The double mutation
T250S/V275A eliminated the AA sensitivity of hIK1. Introducing the
complimentary single amino acid substitutions into rSK2 (S359T and
A384V) conferred partial AA sensitivity to rSK2, 21 ± 3% and
31 ± 3%, respectively. Further, introducing the double mutation
S359T/A384V into rSK2 resulted in a 63 ± 8% (n = 9) inhibition by AA, thereby demonstrating the ability to introduce
this inhibitory AA binding site into another member of the
KCNN gene family. These results demonstrate that AA
interacts with the pore-lining amino acids, Thr250 and
Val275 in hIK1, conferring inhibition of hIK1 by AA and
that AA and clotrimazole share similar, if not identical, molecular
sites of interaction.
Molecular Localization of the Inhibitory Arachidonic Acid Binding
Site to the Pore of hIK1*
, Department of Physiology, University of
Otago, Dunedin, New Zealand and the § Department of
Cell Biology and Physiology, University of Pittsburgh, School of
Medicine, Pittsburgh, Pennsylvania 15261
*
This work was supported by the National Institutes
of Health Grant DK54941 (to D. C. D.), the Lazaro J. Mandel Young
Investigator Award from the American Physiological Society (to
D. C. D.), the University of Otago Dean's Fund (to K. L. H.), and
the University of Otago, Department of Physiology, for sabbatical
support of K. L. H.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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