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Published on: February 4, 2016
Characterization of BK channel splice variants using membrane potential dyes
F Saleem1, I C M Rowe, M J Shipston
1Centre for Integrative Physiology, College of Medicine and Veterinary Medicine, University of Edinburgh, Edinburgh, UK.
Large conductance calcium- and voltage-activated potassium (BK) channels have splice variants with differing sensitivities to calcium. The BK channel blocker paxilline effectively inhibits all variants, making it useful for functional assays.
Area of Science:
- Molecular biology
- Electrophysiology
- Ion channel research
Background:
- Large conductance calcium- and voltage-activated potassium (BK) channels are crucial for cellular excitability.
- Extensive pre-mRNA splicing of the BK channel gene generates diverse functional variants.
- Understanding splice variant-specific properties is key to their physiological roles.
Purpose of the Study:
- To investigate the sensitivity of different BK channel splice variants to intracellular calcium.
- To assess the inhibitory effects of paxilline on these splice variants.
- To explore the utility of membrane potential assays for discriminating BK channel variants.
Main Methods:
- Expression of murine BK channel splice variants (STREX, e22, ZERO) in HEK293 cells.
- Utilized fluorescent membrane potential and patch clamp electrophysiology assays.
- Analyzed responses to ionomycin-induced calcium influx.
- Determined dose-dependent inhibition by paxilline.
Main Results:
- Distinct BK channel splice variants (STREX, e22, ZERO) exhibited varying membrane hyperpolarization in response to calcium influx, with potency order STREX > e22 > ZERO.
- The BK channel inhibitor paxilline showed similar potency (IC50 values) across all tested splice variants.
- IC50 values for paxilline inhibition ranged from 0.35 to 0.70 µmol/L.
Conclusions:
- Membrane potential assays effectively discriminate BK channel splice variants based on calcium sensitivity.
- Paxilline is a potent inhibitor of BK channel splice variants with comparable IC50 values.
- Paxilline can be reliably used in functional assays to inhibit BK channel activity regardless of the specific splice variant.
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