Development of cell-based assay system that utilizes a hyperactive channel mutant for high-throughput screening of BK(Ca) channel modulators

Development of a cell-based functional assay for large-conductance calcium-activated potassium (BK(Ca)) 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 BK(Ca) 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 BK(Ca) 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 BK(Ca) 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 BK(Ca) channel activator. When tested in a commercially available cell-based K⁺ channel assay, cell-lines stably expressing the hyperactive BK(Ca) channel generated a strong fluorescence signal under conditions that are typical for voltage-gated K⁺ channels. In summary, cell-lines expressing the hyperactive mutant BK(Ca) channel represent a new cell-based assay system for investigation of BK(Ca) channels that can be used to screen for novel modulators of these channels.