Recycling of the Ca2+-activated K+ channel, KCa2.3, is dependent upon RME-1, Rab35/EPI64C, and an N-terminal domain

J Biol Chem. 2010 Jun 4;285(23):17938-53. doi: 10.1074/jbc.M109.086553. Epub 2010 Apr 1.


Regulation of the number of Ca(2+)-activated K(+) channels at the endothelial cell surface contributes to control of the endothelium-derived hyperpolarizing factor response, although this process is poorly understood. To address the fate of plasma membrane-localized KCa2.3, we utilized an extracellular epitope-tagged channel in combination with fluorescence and biotinylation techniques in both human embryonic kidney cells and the human microvascular endothelial cell line, HMEC-1. KCa2.3 was internalized from the plasma membrane and degraded with a time constant of 18 h. Cell surface biotinylation demonstrated that KCa2.3 was rapidly endocytosed and recycled back to the plasma membrane. Consistent with recycling, expression of a dominant negative (DN) RME-1 or Rab35 as well as wild type EPI64C, the Rab35 GTPase-activating protein, resulted in accumulation of KCa2.3 in an intracellular compartment. Expression of DN RME-1, DN Rab35, or wild type EPI64C resulted in a decrease in steady-state plasma membrane expression. Knockdown of EPI64C increased cell surface expression of KCa2.3. Furthermore, the effect of EPI64C was dependent upon its GTPase-activating proteins activity. Co-immunoprecipitation studies confirmed an association between KCa2.3 and both Rab35 and RME-1. In contrast to KCa2.3, KCa3.1 was rapidly endocytosed and degraded in an RME-1 and Rab35-independent manner. A series of N-terminal deletions identified a 12-amino acid region, Gly(206)-Pro(217), as being required for the rapid recycling of KCa2.3. Deletion of Gly(206)-Pro(217) had no effect on the association of KCa2.3 with Rab35 but significantly decreased the association with RME-1. These represent the first studies elucidating the mechanisms by which KCa2.3 is maintained at the plasma membrane.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biotinylation
  • Cell Line
  • Cell Membrane / metabolism
  • Electrophysiology / methods
  • Endosomes / metabolism
  • Epitopes / chemistry
  • Gene Deletion
  • Humans
  • Microcirculation
  • Microscopy, Fluorescence / methods
  • Potassium Channels, Calcium-Activated / metabolism*
  • Protein Structure, Tertiary
  • Small-Conductance Calcium-Activated Potassium Channels / metabolism*
  • rab GTP-Binding Proteins / metabolism*


  • Epitopes
  • KCNN3 protein, human
  • Potassium Channels, Calcium-Activated
  • Small-Conductance Calcium-Activated Potassium Channels
  • RAB35 protein, human
  • rab GTP-Binding Proteins