Ten genes with essential structural features of two-pore domain potassium channels were identified in the genome of Drosophila melanogaster. Two Drosophila two-pore domain potassium subunits displayed substantial amino acid similarity to human TWIK-related acid-sensitive K(+) (TASK) channels (38-43%), whereas all others were less than 26% similar to any human homolog. The cDNAs of Drosophila TASK (dTASK)-6 and dTASK-7 channels were isolated from adult fruit flies. In Northern blots dTASK transcripts were found predominantly in the head fraction of adult flies and whole-mount brain in situ hybridizations showed strongly overlapping expression patterns of both dTASK isoforms in the antennal lobes. When heterologously expressed in Drosophila Schneider 2 cells, dTASK-6 gave rise to rapidly activating K(+)-selective currents that steeply depended on external pH. Structural elements in the extracellular M1-P1 loop of dTASK-6 were found to be involved in proton sensation. In contrast to mammalian TASK channels, the pH sensitivity was independent of extracellular histidines adjacent to the GYG selectivity filter (His98). As revealed by mutational analysis, functional expression of dTASK-7 was prevented by two nonconserved amino acids (Ala92-Met93) in the pore domain. When these two residues were replaced by conserved Thr92-Thr93, typical K(+)-selective leak currents were generated that were insensitive to changes in external pH. Nonfunctional wildtype dTASK-7 channels appeared to form heteromeric assemblies with dTASK-6. Following cotransfection of dTASK-6 and wildtype dTASK-7 (or when engineered as concatemers), K(+) currents were observed that were smaller in amplitude, harbored slower activation kinetics and were considerably less inhibited by local anesthetics as compared with dTASK-6. Thus, pore-loop residues in dTASK-7 changed functional and pharmacological properties in heteromeric dTASK channels.