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

• 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.