Conserved Single Residue in the BK Potassium Channel Required for Activation by Alcohol and Intoxication in C. elegans

Abstract

Alcohol directly modulates the BK potassium channel to alter behaviors in species ranging from invertebrates to humans. In the nematode Caenorhabditis elegans, mutations that eliminate the BK channel, SLO-1, convey dramatic resistance to intoxication by ethanol. We hypothesized that certain conserved amino acids are critical for ethanol modulation, but not for basal channel function. To identify such residues, we screened C. elegans strains with different missense mutations in the SLO-1 channel. A strain with the SLO-1 missense mutation T381I in the RCK1 domain was highly resistant to intoxication. This mutation did not interfere with other BK channeldependent behaviors, suggesting that the mutant channel retained normal in vivo function. Knock-in of wild-type versions of the worm or human BK channel rescued intoxication and other BK channel-dependent behaviors in a slo-1-null mutant background. In contrast, knock-in of the worm T381I or equivalent human T352I mutant BK channel selectively rescued BK channel-dependent behaviors while conveying resistance to intoxication. Single-channel patch-clamp recordings confirmed that the human BK channel engineered with the T352I missense mutation was insensitive to activation by ethanol, but otherwise had normal conductance, potassium selectivity, and only subtle differences in voltage dependence. Together, our behavioral and electrophysiological results demonstrate that the T352I mutation selectively disrupts ethanol modulation of the BK channel. The T352I mutation may alter a binding site for ethanol and/or interfere with ethanol-induced conformational changes that are critical for behavioral responses to ethanol.

Mechanisms of Action and Persistent Neuroplasticity by Drugs of Abuse

“…This seems to correlate in mouse models with quicker escalation of limited access alcohol drinking after induction of dependence by chronic intermittent ethanol inhalation in the absence of the b1 subunit (Kreifeldt et al, 2013) and quicker tolerance development to motor impairment in the absence of the b4 subunit . The search for the site of action of ethanol and other n-alcohols on BK channel-forming a subunits is very actively pursued Davis et al, 2014). The discovery of this site of action might allow the rational development of an alcohol antagonist for BK channels.…”

Caenorhabditis elegans as a Model to Study the Molecular and Genetic Mechanisms of Drug Addiction

“…38 The BK potassium channel appears to subserve behavioral responses across multiple species including humans. 32,33,42 Additional work has identified a specific residue (T381I) on the channel that confers dramatic and selective resistance to the behavioral effects of EtOH. 32 Thus, the BK potassium channel is a verified mediator of the effects of EtOH across phyla and may serve as a target for the identification and development of new treatments for alcohol use disorders.…”

Modulation of BK Channels by Ethanol

“…More recent studies using engineered BK channel-forming slo1 proteins (Bukiya et al, 2014) and genetic screening or mutation of the BK channel-coding gene in Caenorhabditis elegans (Slo1) (Davis, Scott, Hu, & Pierce-Shimomura, 2014; Davis et al, 2015) have buttressed the idea of an ethanol-recognition site in the BK channel-forming protein (also termed BK α subunit or slo1 channel protein) by satisfying criteria (2) and (3). While criterion (4) has yet to be met, it should be noted that the Molecular Modeling (MM) and Molecular Dynamics (MD) data from Bukiya et al (2014) were based on crystallographic data of the BK channel cytosolic tail domain (CTD) (wherein the ethanol binding site has been mapped) in both “open” (Ca 2+ i -bound; P.…”