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

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 (BK_Ca) channels. Utilizing human embryonic kidney (HEK293) cells expressing the BK_Ca α subunit, a 96-well cell-based nonradioactive rubidium efflux screen for channel openers and inhibitors was established. Known BK_Ca 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 BK_Ca 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 BK_Ca 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.