Tandem pore domain acid-sensitive K ؉ channel 3 (TASK-3) is a new member of the tandem pore domain potassium channel family. A cDNA encoding a 365-amino acid polypeptide with four putative transmembrane segments and two pore regions was isolated from guinea pig brain. An orthologous sequence was cloned from a human genomic library. Although TASK-3 is 62% identical to TASK-1, the cytosolic C-terminal sequence is only weakly conserved. Analysis of the gene structure identified an intron within the conserved GYG motif of the first pore region. Reverse transcriptase-polymerase chain reaction analysis showed strong expression in brain but very weak mRNA levels in other tissues. Cellattached patch-clamp recordings of TASK-3 expressed in HEK293 cells showed that the single channel currentvoltage relation was inwardly rectifying, and open probability increased markedly with depolarization. Removal of external divalent cations increased the mean single channel current measured at ؊100 mV from ؊2.3 to ؊5.8 pA. Expression of TASK-3 in Xenopus oocytes revealed an outwardly rectifying K ؉ current that was strongly decreased in the presence of lower extracellular pH. Substitution of the histidine residue His-98 by asparagine or tyrosine abolished pH sensitivity. This histidine, which is located at the outer part of the pore adjacent to the selectivity filter, may be an essential component of the extracellular pH sensor.

Diazoxide and 5‐hydroxydecanoate (5‐HD; C10:0) are reputed to target specifically mitochondrial ATP‐sensitive K+ (KATP) channels. Here we describe KATP channel‐independent targets of diazoxide and 5‐HD in the heart. Using submitochondrial particles isolated from pig heart, we found that diazoxide (10‐100 μm) dose‐dependently decreased succinate oxidation without affecting NADH oxidation. Pinacidil, a non‐selective KATP channel opener, did not inhibit succinate oxidation. However, it selectively inhibited NADH oxidation. These direct inhibitory effects of diazoxide and pinacidil cannot be explained by activation of mitochondrial KATP channels. Furthermore, application of either diazoxide (100 μm) or pinacidil (100 μm) did not decrease mitochondrial membrane potential, assessed using TMRE (tetramethylrhodamine ethyl ester), in isolated guinea‐pig ventricular myocytes. We also tested whether 5‐HD, a medium‐chain fatty acid derivative which blocks diazoxide‐induced cardioprotection, was ‘activated’ via acyl‐CoA synthetase (EC 6.2.1.3), an enzyme present both on the outer mitochondrial membrane and in the matrix. Using analytical HPLC and electrospray ionisation mass spectrometry, we showed that 5‐HD‐CoA (5‐hydroxydecanoyl‐CoA) is indeed synthesized from 5‐HD and CoA via acyl‐CoA synthetase. Thus, 5‐HD‐CoA may be the active form of 5‐HD, serving as substrate for (or inhibiting) acyl‐CoA dehydrogenase (β‐oxidation) and/or exerting some other cellular action. In conclusion, we have identified KATP channel‐independent targets of 5‐HD, diazoxide and pinacidil. Our findings question the assumption that sensitivity to diazoxide and 5‐HD implies involvement of mitochondrial KATP channels. We propose that pharmacological preconditioning may be reelated to partial inhibition of respiratory chain complexes.

The function of the heart depends critically on an adequate oxygen supply through the coronary arteries. Coronary arteries dilate when the intravascular oxygen tension decreases. Hypoxic vasodilation in isolated, perfused guinea pig hearts can be prevented by glibenclamide, a blocker of adenosine triphosphate (ATP)-sensitive potassium channels, and can be mimicked by cromakalim, which opens ATP-sensitive potassium channels. Opening of potassium channels in coronary smooth muscle cells and the subsequent drop in intracellular calcium is probably the major cause of hypoxic and ischemic vasodilation in the mammalian heart.

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Two cDNAs encoding novel K؉ channels, THIK-1 and THIK-2 (tandem pore domain halothane inhibited K ؉ channel), were isolated from rat brain. The proteins of 405 and 430 amino acids were 58% identical to each other. Homology analysis showed that the novel channels form a separate subfamily among tandem pore domain K ؉ channels. The genes of the human orthologs were identified as human genomic data base entries. They possess one intron each and were assigned to chromosomal region 14q24.1-14q24.3 (human (h) THIK-1) and 2p22-2p21 (hTHIK-2). In rat (r), THIK-1 (rTHIK-1) is expressed ubiquitously; rTHIK-2 expression was found in several tissues including brain and kidney. In situ hybridization of brain slices showed that rTHIK-2 is strongly expressed in most brain regions, whereas rTHIK-1 expression is more restricted. Heterologous expression of rTHIK-1 in Xenopus oocytes revealed a K ؉ channel displaying weak inward rectification in symmetrical K ؉ solution. The current was enhanced by arachidonic acid and inhibited by halothane. rTHIK-2 did not functionally express. Confocal microscopy of oocytes injected with green fluorescent protein-tagged rTHIK-1 or rTHIK-2 showed that both channel subunits are targeted to the outer membrane. However, coinjection of rTHIK-2 did not affect the currents induced by rTHIK-1, indicating that the two channel subunits do not form heteromers.