A Novel Approach for Locating the Sites of Drug Action on BK Channels

Abstract

High-conductance Ca(2+)-and voltage-activated K(+) (Slo1 or BK) channels play key roles in many normal and pathophysiological processes, such as sinoatrial node pacing, blood pressure regulation, neurotransmitter release, autism, and epilepsy. The Slo1 channel can be divided into a trans-membrane core module comprised of the pore gate domain (PGD) surrounded by four voltage sensor (VS domains), and a large cytosolic domain (CTD) which forms an intracellular gating ring. DHA (docosahexaenoic acid), protons, and DiBAC4(3) (bis-(1,3-dibutylbarbituric acid) trimethine oxonol) can activate wt BK channels. Our construct of functional Slo1-core channels comprised of the PGD and VS domains without the CTD provides a tool to explore drug target domains of BK channels. In this study, we test the action of DHA, protons, and DiBac4(3) on wt and Slo1-core channels using single-channel and macro-patch recordings to measure channel activity before and after application of activating agents. We found that both DHA and DiBAC4(3) activate Slo1-core channels. This was the case with or without beta1 subunits. These results indicate that DHA and DiBAC4(3) do not require the CTD to activate Slo1 channels. We also found that protons did not activate Slo1-core channels, which directly shows that protons require the CTD for their action, while having little or no direct action on the core of the channel. Our results for DHA and protons are consistent with previous studies using less direct experimental approaches. Our observation that DiBAC4(3) can activate core channels in the absence of the gating ring is a novel. This study is a clear demonstration of the utility of the functional Slo1-core constructs we have developed for locating the sites of action of BK channel enhancers and modulators.