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J. Biol. Chem., Vol. 278, Issue 10, 8476-8486, March 7, 2003
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From the Department of Cell Biology and Physiology, University of
Pittsburgh, Pittsburgh, Pennsylvania 15261
We demonstrate that the C-terminal truncation of
hIK1 results in a loss of functional channels. This could be caused by
either (i) a failure of the channel to traffic to the plasma membrane or (ii) the expression of non-functional channels. To delineate among
these possibilities, a hemagglutinin epitope was inserted into the
extracellular loop between transmembrane domains S3 and S4. Surface
expression and channel function were measured by immunofluorescence, cell surface immunoprecipitation, and whole-cell patch clamp
techniques. Although deletion of the last 14 amino acids of hIK1
(L414STOP) had no effect on plasma membrane expression and function,
deletion of the last 26 amino acids (K402STOP) resulted in a complete
loss of membrane expression. Mutation of the leucine heptad repeat ending at Leu406 (L399A/L406A) completely
abrogated membrane localization. Additional mutations within the heptad
repeat (L385A/L392A, L392A/L406A) or of the a positions
(I396A/L403A) resulted in a near-complete loss of membrane-localized
channel. In contrast, mutating individual leucines did not compromise
channel trafficking or function. Both membrane localization and
function of L399A/L406A could be partially restored by incubation at
27 °C. Co-immunoprecipitation studies demonstrated that leucine
zipper mutations do not compromise multimer formation. In contrast, we
demonstrated that the leucine zipper region of hIK1 is capable of
co-assembly and that this is dependent upon an intact leucine zipper.
Finally, this leucine zipper is conserved in another member of the gene
family, SK3. However, mutation of the leucine zipper in SK3 had
no effect on plasma membrane localization or function. In conclusion,
we demonstrate that the C-terminal leucine zipper is critical to
facilitate correct folding and plasma membrane trafficking of hIK1,
whereas this function is not conserved in other gene family members.
Trafficking of the Ca2+-activated K+
Channel, hIK1, Is Dependent upon a C-terminal Leucine Zipper*
*
This work was supported by the Lazaro J. Mandel Young
Investigator award from the American Physiological Society (to
D. C. D.), National Institutes of Health Grant DK54941 (to
D. C. D.), and the Pennsylvania/Delaware Affiliate of the American
Heart Association Grant 0120544U (to C. A. S.).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.
To whom correspondence should be addressed: Dept. of Cell Biology
and Physiology, University of Pittsburgh School of Medicine, S312 BST,
3500 Terrace St., Pittsburgh, PA 15261. Tel.: 412-383-8755; Fax:
412-648-8330; E-mail: dd2@pitt.edu.
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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