Döring Frank, Scholz Henrike, Kühnlein Ronald P, Karschin Andreas, Wischmeyer Erhard
Institute of Physiology, University of Würzburg, Röntgenring 9, 97070 Würzburg, Germany.
Eur J Neurosci. 2006 Oct;24(8):2264-74. doi: 10.1111/j.1460-9568.2006.05102.x.
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.
在黑腹果蝇基因组中鉴定出了具有双孔结构域钾通道基本结构特征的10个基因。两个果蝇双孔结构域钾亚基与人类TWIK相关酸敏感钾（TASK）通道显示出相当大的氨基酸相似性（38 - 43%），而其他所有亚基与任何人类同源物的相似性均低于26%。从成年果蝇中分离出了果蝇TASK（dTASK）-6和dTASK-7通道的cDNA。在Northern印迹中，dTASK转录本主要在成年果蝇的头部组分中被发现，而全脑原位杂交显示两种dTASK同工型在触角叶中具有强烈重叠的表达模式。当在果蝇Schneider 2细胞中异源表达时，dTASK-6产生快速激活的钾选择性电流，该电流强烈依赖于细胞外pH。发现dTASK-6细胞外M1-P1环中的结构元件参与质子感知。与哺乳动物TASK通道不同，pH敏感性不依赖于与GYG选择性过滤器相邻的细胞外组氨酸（His98）。如突变分析所示，dTASK-7的功能表达被孔结构域中的两个非保守氨基酸（Ala92 - Met93）所阻止。当这两个残基被保守的Thr92 - Thr93取代时，产生了典型的钾选择性泄漏电流，该电流对细胞外pH的变化不敏感。无功能的野生型dTASK-7通道似乎与dTASK-6形成异源多聚体组装体。在共转染dTASK-6和野生型dTASK-7后（或当构建为串联体时），观察到钾电流，其幅度较小，具有较慢的激活动力学，并且与dTASK-6相比，对局部麻醉剂的抑制作用明显较小。因此，dTASK-7中的孔环残基改变了异源多聚体dTASK通道的功能和药理学特性。
