Functional Analysis of Large Conductance Ca2+-Activated K+ Channels: Ion Flux Studies by Atomic Absorption Spectrometry

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Abstract
Although techniques such as 86Rb+ flux provide a sensitive measure of K+ channel activity, the relatively short half-life and high-energy emission, together with the quantities of radioactive material generated, hinder the usefulness of flux-based formats in high throughput screening efforts. This study elaborates on the utilization of flame atomic absorption spectrometry (AAS) techniques for a nonradioactive rubidium efflux assay for large conductance Ca2+-activated K+ channels (BKCa) channels. Utilizing human embryonic kidney (HEK293) cells expressing the BKCa α subunit, a 96-well cell-based nonradioactive rubidium efflux screen for channel openers and inhibitors was established. Known BKCa channel openers, including NS1608, NS1619, and NS-8, activated rubidium efflux with EC50 values ranging from 1 to 4 μM in both radioactive and nonradioactive efflux formats. Compounds such as iberiotoxin, paxilline, and charybdotoxin inhibited rubidium efflux responses evoked by the BKCa channel opener NS1608 in both radioactive and nonradioactive efflux formats. The IC50 values of the inhibitors in AAS format were comparable to those derived from 86Rb+ efflux assays. The present studies show that the pharmacological profiles of BKCa channels assessed by AAS compare well with those obtained using the 86Rb+ efflux assay, and support the utility of nonradioactive efflux format for higher throughput screening campaigns for novel K+ channel modulators.