Development of alcohol abuse has a strong genetic component. People who are innately resistant to intoxication are at high risk to abuse alcohol and vice versa. This genetic predisposition is linked to variation in genes that encode targets of alcohol or their downstream effectors involved in intoxication. Genetic screens in C. elegans previously determined that the gene
slo-1 represents a central target for alcohol intoxication in the worm.
slo-1 encodes the highly conserved large-conductance, calcium-activated potassium (BK) channel. The BK channel has emerged as a key mediator for intoxication and tolerance across species from worm, fly, mouse to human. At the physiological level, BK channel activity is modulated by clinically relevant concentrations of ethanol (~20mM). To reveal specific residues in the BK channel that are required for activation by alcohol, and thus may influence sensitivity to behavioral intoxication, we are performing multiple genetic screens. First, we are screening
slo-1 mutants from the million mutation project. Second, we are performing site-directed mutagenesis on the worm and human BK channels. We are able to study the human BK channel in the worm because we rescued ethanol sensitivity in a
slo-1 null worm by expression of hslo. Novel mutants that are resistant to intoxication will either contain 1) a null mutation in
slo-1 that provides no new information, or 2) a novel non-null mutation that provides insight into how specific residues on the BK channel interact with alcohol, and how this interaction alters intoxication. To distinguish non-null candidates, the locomotor posture of mutants recovered from the genetic screens will be compared against wild-type and a known
slo-1 null mutant. Candidate non-null mutants will display different locomotor posture than the known
slo-1 null mutant. Currently, we have isolated multiple candidate non-null mutants with resistance to intoxication. Further analysis using in vivo patch-clamp recordings will assess how specific mutations alter basal BK channel function and the response to alcohol.