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Development of cell-based assay system that utilizes a hyperactive channel mutant for high-throughput screening of BKCa channel modulators

Paper ID Volume ID Publish Year Pages File Format Full-Text
23543 43451 2013 6 PDF Available
Title
Development of cell-based assay system that utilizes a hyperactive channel mutant for high-throughput screening of BKCa channel modulators
Abstract

•A hyper-active of BKCa channel was designed by a double mutation (803D/N806K) and successfully expressed.•The mutant channel is fully activated by physiological voltages under resting intracellular Ca2+.•Mammalian cells stably expressing G803D/N806K channels generate robust fluorescent signals in a fluorescence-based cell assay.•The cell-based assay system utilizing G803D/N806K can be applied for high throughput screening of novel modulators of BKCa channel.

Development of a cell-based functional assay for large-conductance calcium-activated potassium (BKCa) channels is challenging because of the unique requirement of both voltage and high concentrations of Ca2+ for activation of these channels. Here, we describe a new cell-based assay system that utilizes a hyperactive mutant BKCa channel. The hyperactive mutant was generated by introducing two point-mutations into the cytosolic flexible interface between the two RCK domains of the wild-type BKCa channel. The mutant channel exhibited a large negative shift in its conductance–voltage relationship, which indicates activation by modest depolarization at resting concentrations of intracellular Ca2+. Unlike the wild-type BKCa channel, the hyperactive mutant did not require a concomitant increase of intracellular Ca2+ for activation. Despite the observed shift in its voltage activation profile, activity of the mutant channel was further potentiated by a known BKCa channel activator. When tested in a commercially available cell-based K+ channel assay, cell-lines stably expressing the hyperactive BKCa channel generated a strong fluorescence signal under conditions that are typical for voltage-gated K+ channels. In summary, cell-lines expressing the hyperactive mutant BKCa channel represent a new cell-based assay system for investigation of BKCa channels that can be used to screen for novel modulators of these channels.

Keywords
BKCa, large-conductance calcium-activated potassium (channel); RCK, regulator of K+ conductance; RMP, resting membrane potential; WT, wild-typeHigh throughput screening; BKCa channel; Cell-based assay
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Development of cell-based assay system that utilizes a hyperactive channel mutant for high-throughput screening of BKCa channel modulators
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 167, Issue 1, 10 August 2013, Pages 41–46
Authors
, , , , , ,
Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us