Immunofluorescence-based assay to identify modulators of the number of plasma membrane KCa3.1 channels.
BACKGROUND
Intermediate conductance Ca2+-dependent K+ channels (KCa3.1) have been proposed as therapeutic targets for numerous diseases. We recently characterized the endocytic fate of these channels; leading to the possibility that this can be pharmacologically manipulated, thereby altering the number of channels (N) at the plasma membrane.


CONCLUSIONS
We demonstrate that plasma membrane-localized KCa3.1 can be rapidly(10 min) tagged with a fluorophore using a combination of a biotin ligase (BirA) acceptor peptide-tagged channel and an ER-localized BirA. Endocytosis of KCa3.1 was quantified using a 96-well plate format, demonstrating that the ubiquitin-activating enzyme E1 inhibitor UBEI-41, blocks the endocytosis of KCa3.1.


CONCLUSIONS
We describe a novel method for identifying modulators of KCa endocytosis and demonstrate this can be used to modulate Nat the plasma membrane. It is anticipated that altering N will provide novel therapeutic strategies for targeting these channels in disease.
Balut
Corina M
CM
Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, PA 15261, USA.
Gao
Yajuan
Y
Luke
Cliff
C
Devor
Daniel C
DC
D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D004705:Endocytosis; D005455:Fluorescent Antibody Technique / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels / Q000032:analysis* / Q000378:metabolism*; D044764:Ubiquitin-Activating Enzymes / Q000037:antagonists & inhibitors
D005456:Fluorescent Dyes; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; D044764:Ubiquitin-Activating Enzymes