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. RESULTS & DISCUSSION: 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.
CONCLUSION: 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.