A Novel High-Throughput Screening Assay for State-Dependent and Subunit-Dependent BK Channel Modulators

Abstract

BK potassium channels are attractive drug targets for a wide variety of human disorders affecting almost every organ system. However, the therapeutic potential of many BK channel modulators is limited by lack of tissue specificity, reflecting that the pore forming a-subunit (Slo1) is encoded by a single gene. Properties of modulators that can potentially enhance tissue specificity include (a) sensitivity to regulatory subunits that are expressed in a tissue-dependent manner and (b) state-dependent action, conferring sensitivity to tissue-specific differences in membrane voltage and Ca2+ that activate BK channels. We have developed a novel 384-well fluorescent thallium-flux kinetic (FLIPR) assay to identify modulators with these properties. The screen includes assays on a panel of BK channel variants composed of hyperactive human Slo1 mutants (F380Y or R275C) in the presence or absence of different regulatory subunits (β1, β2, β2αFIW, β4, γ1), expressed in U2OS cells with Bacmam virus. Hyperactive Slo1 mutants allow both inhibitors and activators to be reliably detected under resting cellular conditions. Comparison of R275C and F380Y identifies state-dependent modulators since the voltage-sensors of R275C are constitutively activated while F380Y are not. A test screen of small molecules and arachnid venoms revealed a surprising degree of variant-selectivity and identified novel modulators with activities that depend on voltage-sensor activation and/or regulatory subunit expression. Thus, the screen detects modulators that would fail to be detected by conventional screens while also providing information about mechanism of action that should facilitate SAR analysis and development of potent tissue-selective modulators.