Characterization of BK channel splice variants using membrane potential dyes.
OBJECTIVE
Large conductance calcium- and voltage-activated potassium (BK) channels are encoded by a single gene that displays extensive pre-mRNA splicing. Here we exploited a membrane potential assay to investigate the sensitivity of different BK splice variants to elevations in intracellular free calcium and their inhibition by the BK channel blocker paxilline.


METHODS
Murine BK channel splice variants were expressed in human embryonic kidney 293 cells and their properties analysed in response to ionomycin-induced calcium influx in both fluorescent membrane potential (fluorescent-imaging plate reader) and patch clamp electrophysiological assays. The dose-dependent inhibition of distinct splice variants by the BK channel-specific blocker paxilline was also investigated.


RESULTS
Ionomycin-induced calcium influx induced a robust hyperpolarization of human embryonic kidney 293 cells expressing distinct BK channel splice variants: stress regulated exon (STREX), e22 and ZERO. Splice variant expression resulted in membrane hyperpolarization that displayed a rank order of potency in response to calcium influx of STREX > e22 > ZERO. The BK channel inhibitor paxilline exhibited very similar potency on all three splice variants with IC(50)s in membrane potential assays of 0.35 +/- 0.04, 0.37 +/- 0.03 and 0.70 +/- 0.02 micromol x L(-1) for STREX, ZERO and e22 respectively.


CONCLUSIONS
BK channel splice variants can be rapidly discriminated using membrane potential based assays, based on their sensitivity to calcium. BK channel splice variants are inhibited by the specific blocker paxilline with similar IC(50)s. Thus, paxilline may be used in functional assays to inhibit BK channel function, irrespective of the variant expressed.
Saleem
F
F
Centre for Integrative Physiology, College of Medicine and Veterinary Medicine, University of Edinburgh, Edinburgh, UK.
Rowe
I C M
IC
Shipston
M J
MJ
D017398:Alternative Splicing; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D005091:Exons; D005456:Fluorescent Dyes; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D015759:Ionomycin / Q000494:pharmacology; D051036:Large-Conductance Calcium-Activated Potassium Channels / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D008564:Membrane Potentials; D018408:Patch-Clamp Techniques; D026902:Potassium Channel Blockers / Q000494:pharmacology; D014162:Transfection
D005456:Fluorescent Dyes; D007211:Indoles; D051036:Large-Conductance Calcium-Activated Potassium Channels; D026902:Potassium Channel Blockers; C048220:paxilline; D015759:Ionomycin; D002118:Calcium