Ethanol is a widely used and abused drug, but the molecular mechanisms that drive its acute behavioral effects are not understood. Ethanol exposure alters the behavior of invertebrates and mammals, suggesting the existence of conserved ethanol targets in the nervous system. Genetic studies of ethanol action on locomotion in C. elegans have identified Slo-1 as a potential target of ethanol action (Davies 2003 Cell 115:655). Slo-1 has been shown to encode a calcium-activated, large-conductance, potassium-selective channel called BKca. When the
slo-1 gene was deleted from neurons, the animals were resistant to ethanol concentrations up to 500 mM. Other genetic studies in mice and Drosophila have also identified the Gs-cAMP-PKA signaling system as an antagonist of ethanol action. Previous unpublished work in our laboratory had demonstrated that the S/T protein phosphatase, PP1 is required for the effects of ethanol on recombinant mammalian BKca activity. To further study the molecular mechanisms of Slo-1 potassium channel in response to ethanol in C. elegans, we have expressed the C. elegans
slo-1A gene in a heterologous system. It behaved like the mammalian ZERO isoform in that it was stimulated by PKA and inhibited by ethanol. Motif Scan software identifies several putative PKA phosphorylation sites in the Slo-1A protein, and site-directed mutagenesis of S744 eliminated the response to ethanol. Although the effects of ethanol on wild type channel activity are significant, they are fairly small, ~37% change in peak current. Nevertheless, mutation of S744 had dramatic effects on the worms'' behavioral sensitivity to ethanol. As reported previously, the
slo-1 knockout animals were impervious to ethanol. However, expression of the wild type construct that we had characterized electrophysiologically fully restored the effect of alcohol on the worms'' locomotor activity. In contrast, the worms that express the mutant S744A
slo-1 protein remain impervious to ethanol. Similarly, inhibition of neuronal protein phosphatase 1 activity by overexpression of a mammalian inhibitor 2 protein made the worms impervious to ethanol at 500 mM, suggesting the PP1 activity is also important for ethanol response in C. elegans. Thus, we hypothesize that PP1 is the molecular target of ethanol in the brain and future studies will examine the mechanism of this effect and its relevance to other channels that are regulated by ethanol in the mammalian central nervous system.