Recycling of the Ca2+-activated K+ channel, KCa2.3, is dependent upon RME-1, Rab35/EPI64C, and an N-terminal domain.
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.
Gao
Yajuan
Y
Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
Balut
Corina M
CM
Bailey
Mark A
MA
Patino-Lopez
Genaro
G
Shaw
Stephen
S
Devor
Daniel C
DC
D019921:Biotinylation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004594:Electrophysiology / Q000379:methods; D011992:Endosomes / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry; D017353:Gene Deletion; D006801:Humans; D008833:Microcirculation; D008856:Microscopy, Fluorescence / Q000379:methods; D024681:Potassium Channels, Calcium-Activated / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051640:Small-Conductance Calcium-Activated Potassium Channels / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000378:metabolism*
D000939:Epitopes; C406328:KCNN3 protein, human; D024681:Potassium Channels, Calcium-Activated; D051640:Small-Conductance Calcium-Activated Potassium Channels; C511183:RAB35 protein, human; D020691:rab GTP-Binding Proteins