Large conductance, Ca++-activated K+ channel (BK) channel activity, involved in the regulation of arterial tone and central neuron excitability, has been found to be modulated by acute ethanol (EtOH) at relevant concentrations. To understand the mechanisms which govern the interaction of EtOH with specific regions in its targets, in particular ion channel proteins, is critical in the alcohol field since, with a few recent exceptions, the specific interaction of EtOH at relevant concentrations with selective regions of a protein, a requirement for a """"""""receptor"""""""" from classical pharmacology, has remained largely elusive. We have found that BK channel activity is not potentiated by EtOH in arterial smooth muscle, while it markedly increased in BK channels from nerve terminals and PC12 cells. This dichotomy remained when EtOH action was studied on two 99% identical cloned channels encoded by slo genes, inserted in the same proteo-lipid environment: 50 mM EtOH inhibits channels from arterial smooth muscle (bslo alpha subunit) whereas it activates channels from brain (mslo, alpha subunit) when both are expressed in oocytes. Thus, the main hypothesis is that the differential action of EtOH on these channels is due to specific differences in the sequences between the two channel proteins. Using single channel recordings from cell-free patches, the concentration-dependence of EtOH action on bslo channels, and the channel properties modified by EtOH, will be determined. Results will be compared to those from mslo channels. Since channel properties are linked to defined regions in the proteins, differential EtOH-modification EtOH-modification of specific properties in the two clones will direct us to defined regions in the protein as putative recognition sites for EtOH. Then, the study of EtOH action on electrophysiological properties of mutated channels constructed by exchanging non-conserved regions of these two proteins encoded by slo genes will confirm our predictions of which regions in the protein determine sensitivity to EtOH and underlie its differential effect. Site- directed mutagenesis in non-conserved regions between the two clones will address which amino acids are involved in the BK channel-mutagenesis in non-conserved regions between the two clones will address which amino acids are involved in the Bk channel-EtOH interaction. Results from BK clones will help us to focus on specific channel regions and properties targeted by EtOH when the action of the drug is evaluated on BK channels in cerebral arterial cells, where the drug produces direct vasoconstriction, probably due to an inhibition of BK channels. Elucidating the molecular mechanisms underlying the interaction between EtOH and BK channels from arteries will help in understanding direct actions of the drug on arterial tone, and, perhaps, lead to development of clinically useful agents.
