Trafficking of the Ca2+-activated K+Channel, hIK1, Is Dependent upon a C-terminal Leucine Zipper

• Published in: The Journal of biological chemistry Vol. 278; no. 10; pp. 8476 - 8486
• Main Authors: Syme, Colin A, Hamilton, Kirk L, Jones, Heather M, Gerlach, Aaron C, Giltinan, LeeAnn, Papworth, Glenn D, Watkins, Simon C, Bradbury, Neil A, Devor, Daniel C
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 07-03-2003
• Subjects: Base Sequence; Blotting, Western; Cell Line; Cell Membrane - metabolism; DNA Primers; Fluorescent Antibody Technique; Humans; Intermediate-Conductance Calcium-Activated Potassium Channels; Leucine Zippers; Mutagenesis; Potassium Channels - metabolism; Potassium Channels, Calcium-Activated; Protein Transport; Temperature
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M210072200

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 Leu 406 (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.