Trafficking of the Ca2+-activated K+ channel, hIK1, is dependent upon a C-terminal leucine zipper

J Biol Chem. 2003 Mar 7;278(10):8476-86. doi: 10.1074/jbc.M210072200. Epub 2002 Dec 18.

Abstract

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 degrees 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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 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

Substances

  • DNA Primers
  • Intermediate-Conductance Calcium-Activated Potassium Channels
  • KCNN4 protein, human
  • Potassium Channels
  • Potassium Channels, Calcium-Activated