Calcium-activated potassium channels mediate many biologically important functions in electrically excitable cells. Despite recent progress in the molecular analysis of voltage-activated K+ channels, Ca(2+)-activated K+ channels have not been similarly characterized. The Drosophila slowpoke (slo) locus, mutations of which specifically abolish a Ca(2+)-activated K+ current in muscles and neurons, provides an opportunity for molecular characterization of these channels. Genomic and complementary DNA clones from the slo locus were isolated and sequenced. The polypeptide predicted by slo is similar to voltage-activated K+ channel polypeptides in discrete domains known to be essential for function. Thus, these results indicate that slo encodes a structural component of Ca(2+)-activated K+ channels.

Rapid tolerance, measured as a reduction in the duration of sedation, is a pharmacokinetic response to ethanol that does not occur without slowpoke expression in the nervous system in Drosophila. The slowpoke channel must be involved in triggering or producing a homeostatic mechanism that opposes the sedative effects of ethanol.

Changes in neural activity caused by exposure to drugs may trigger homeostatic mechanisms that attempt to restore normal neural excitability. In Drosophila, a single sedation with the anesthetic benzyl alcohol changes the expression of the slo K ؉ channel gene and induces rapid drug tolerance. We demonstrate linkage between these two phenomena by using a mutation and a transgene. A mutation that eliminates slo expression prevents tolerance, whereas expression from an inducible slo transgene mimics tolerance in naïve animals. The behavioral response to benzyl alcohol can be separated into an initial phase of hyperkinesis and a subsequent phase of sedation. The hyperkinetic phase causes a drop in slo gene expression and makes animals more sensitive to benzyl alcohol. It is the sedative phase that stimulates slo gene expression and induces tolerance. We demonstrate that the expression level of slo is a predictor of drug sensitivity.drug abuse ͉ potassium channel ͉ transcription regulation

In Drosophila, ethanol sedation induces slowpoke expression in the nervous system and results in ethanol tolerance. The induction of slowpoke expression alone is sufficient to produce a phenotype that is indistinguishable from true ethanol tolerance. Therefore, the regulation of the slowpoke BK-type channel gene must play an integral role in the Drosophila ethanol response.

Tolerance to drugs that affect neural activity is mediated, in part, by adaptive mechanisms that attempt to restore normal neural excitability. Changes in the expression of ion channel genes are thought to play an important role in these neural adaptations. The slo gene encodes the pore-forming subunit of BK-type Ca2+-activated K+ channels, which regulate many aspects of neural activity. Given that induction of slo gene expression plays an important role in the acquisition of tolerance to sedating drugs, we investigated the molecular mechanism of gene induction. Using chromatin immunoprecipitation followed by real-time PCR, we show that a single brief sedation with the anesthetic benzyl alcohol generates a spatiotemporal pattern of histone H4 acetylation across the slo promoter region. Inducing histone acetylation with a histone deacetylase inhibitor yields a similar pattern of changes in histone acetylation, up-regulates slo expression, and phenocopies tolerance in a slo-dependent manner. The cAMP response element binding protein (CREB) is an important transcription factor mediating experience-based neuroadaptations. The slo promoter region contains putative binding sites for the CREB transcription factor. Chromatin immunoprecipitation assays show that benzyl alcohol sedation enhances CREB binding within the slo promoter region. Furthermore, activation of a CREB dominant-negative transgene blocks benzyl alcohol–induced changes in histone acetylation within the slo promoter region, slo induction, and behavioral tolerance caused by benzyl alcohol sedation. These findings provide unique evidence that links molecular epigenetic histone modifications and transcriptional induction of an ion channel gene with a single behavioral event.

The Drosophila slowpoke (slo) gene encodes a subunit of a CAK channel homologous to the vertebrate BK channel. We have examined slo expression throughout development. It is expressed in muscle cells, neurons of the CNS and PNS, mushroom bodies, a limited number of cells in embryonic and larval midgut and in epithelial-derived tracheal cells. The promoter has been cloned and shown to direct expression in the same pattern as the endogenous gene in both neural and epithelial-derived cells. During pupariation and embryogenesis, slo is expressed in muscles many hours prior to the appearance of functional channels.

Sustained or repeated exposure to sedating drugs, such as alcohol, triggers homeostatic adaptations in the brain that lead to the development of drug tolerance and dependence. These adaptations involve long-term changes in the transcription of drug-responsive genes as well as an epigenetic restructuring of chromosomal regions that is thought to signal and maintain the altered transcriptional state. Alcohol-induced epigenetic changes have been shown to be important in the long-term adaptation that leads to alcohol tolerance and dependence endophenotypes. A major constraint impeding progress is that alcohol produces a surfeit of changes in gene expression, most of which may not make any meaningful contribution to the ethanol response under study. Here we used a novel genomic epigenetic approach to find genes relevant for functional alcohol tolerance by exploiting the commonalities of two chemically distinct alcohols. In Drosophila melanogaster, ethanol and benzyl alcohol induce mutual cross-tolerance, indicating that they share a common mechanism for producing tolerance. We surveyed the genome-wide changes in histone acetylation that occur in response to these drugs. Each drug induces modifications in a large number of genes. The genes that respond similarly to either treatment, however, represent a subgroup enriched for genes important for the common tolerance response. Genes were functionally tested for behavioral tolerance to the sedative effects of ethanol and benzyl alcohol using mutant and inducible RNAi stocks. We identified a network of genes that are essential for the development of tolerance to sedation by alcohol.

Invertebrate species possess one or two Na ؉ channel genes, yet there are 10 in mammals. When did this explosive growth come about during vertebrate evolution? All mammalian Na ؉ channel genes reside on four chromosomes. It has been suggested that this came about by multiple duplications of an ancestral chromosome with a single Na ؉ channel gene followed by tandem duplications of Na ؉ channel genes on some of these chromosomes. Because a large-scale expansion of the vertebrate genome likely occurred before the divergence of teleosts and tetrapods, we tested this hypothesis by cloning Na ؉ channel genes in a teleost fish. Using an approach designed to clone all of the Na ؉ channel genes in a genome, we found six Na ؉ channel genes. Phylogenetic comparisons show that each teleost gene is orthologous to a Na ؉ channel gene or gene cluster on a different mammalian chromosome, supporting the hypothesis that four Na ؉ channel genes were present in the ancestors of teleosts and tetrapods. Further duplications occurred independently in the teleost and tetrapod lineages, with a greater number of duplications in tetrapods. This pattern has implications for the evolution of function and specialization of Na ؉ channel genes in vertebrates. Sodium channel genes also are linked to homeobox (Hox) gene clusters in mammals. Using our phylogeny of Na ؉ channel genes to independently test between two models of Hox gene evolution, we support the hypothesis that Hox gene clusters evolved as (AB) (CD) rather than {D[A(BC)]}.