Elucidating residues on the BK channel required for activation by alcohol and intoxication in C. elegans
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Alcohol produces changes in behavior through molecular effects on ion channels, enzymes and transporters. Many proteins have been elucidated that at least in part mediate behavioral changes induced by alcohol. However, it has been difficult thus far to uncover key amino acid residues within a protein that are necessary for the effects of alcohol. This information is critical, potentially leading to effective pharmacological treatments for alcohol use disorders (AUD) and identification of allelic variations that predispose an individual for AUD. The big conductance voltage- and calcium-activated potassium (BK) channel has recently emerged as a critical protein for the effects of alcohol across species. In this dissertation, we study the molecular action of alcohol on the BK channel, and how this action contributes to behavioral intoxication. To accomplish this, we first provide credence for using the nematode C. elegans for studying the behavioral effects of ethanol. We demonstrate how behavioral intoxication and internal ethanol concentration in C. elegans is altered by the osmolarity of the ethanol-solution, reconciling results from previous conflicting reports in the literature. We then identify the amino acid residue T381 on the BK channel in C. elegans is critical for behavioral intoxication, but not other BK channel-dependent behaviors. These results suggest a functional BK channel resistant to ethanol. By knocking-in the human BK channel, we then demonstrate that the equivalent residue, T352 is also critical for behavioral intoxication in C. elegans, but not other BK channel-dependent behaviors. Using single-channel recordings, we find that the T352 residue is critical for the potentiating effects of ethanol on the human BK channel, without being critical for basal-function. Finally, we investigate the role of calcium-sensing residues on the worm BK channel for behavioral intoxication in C. elegans. We find that these residues are non-essential for intoxication, in contrast to in vitro reports in the mammalian channel suggesting the calcium-sensing residues are critical for ethanol-activation of the BK channel.