title: Development of cell-based assay system that utilizes a hyperactive channel mutant for high-throughput screening of BK(Ca) channel modulators.
abstract: 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.
authors.affiliation: School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea.
authors.forename: Byoung-Cheol
authors.initials: BC
authors.lastname: Lee
authors.forename: Hyun-Ju
authors.initials: HJ
authors.lastname: Kim
authors.forename: Sun Hwa
authors.initials: SH
authors.lastname: Park
authors.forename: Tam Thi Thanh
authors.initials: TT
authors.lastname: Phuong
authors.forename: Tong Mook
authors.initials: TM
authors.lastname: Kang
authors.forename: Chul-Seung
authors.initials: CS
authors.lastname: Park
mesh_terms: D045744:Cell Line, Tumor; D005453:Fluorescence; D057166:High-Throughput Screening Assays; D006801:Humans; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits / Q000502:physiology*; D017354:Point Mutation
chemical_list: D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits
keywords: BK(Ca); BK(Ca) channel; Cell-based assay; High throughput screening; RCK; RMP; WT; large-conductance calcium-activated potassium (channel); regulator of K(+) conductance; resting membrane potential; wild-type