Jürgen Daut

KATP channel‐independent targets of diazoxide and 5‐hydroxydecanoate in the heart

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.

Heteromerization of Kir2.x potassium channels contributes to the phenotype of Andersen's syndrome.

Andersens syndrome, an autosomal dominant disorder related to mutations of the potassium channel Kir2.1, is characterized by cardiac arrhythmias, periodic paralysis, and dysmorphic bone structure. The aim of our study was to find out whether heteromerization of Kir2.1 channels with wild-type Kir2.2 and Kir2.3 channels contributes to the phenotype of Andersens syndrome. The following results show that Kir2.x channels can form functional heteromers: (i) HEK293 cells transfected with Kir2.x–Kir2.y concatemers expressed inwardly rectifying K+ channels with a conductance of 28–30 pS. (ii) Expression of Kir2.x–Kir2.y concatemers in Xenopus oocytes produced inwardly rectifying, Ba2+ sensitive currents. (iii) When Kir2.1 and Kir2.2 channels were coexpressed in Xenopus oocytes the IC50 for Ba2+ block of the inward rectifier current differed substantially from the value expected for independent expression of homomeric channels. (iv) Coexpression of nonfunctional Kir2.x constructs, in which the GYG region of the pore region was replaced by AAA, with wild-type Kir2.x channels produced both homomeric and heteromeric dominant-negative effects. (v) Kir2.1 and Kir2.3 channels could be coimmunoprecipitated in membrane extracts from isolated guinea pig cardiomyocytes. (vi) Yeast two-hybrid analysis showed interaction between the N- and C-terminal intracellular domains of different Kir2.x subunits. Coexpression of Kir2.1 mutants related to Andersens syndrome with wild-type Kir2.x channels showed a dominant negative effect, the extent of which varied between different mutants. Our results suggest that differential tetramerization of the mutant allele of Kir2.1 with wild-type Kir2.1, Kir2.2, and Kir2.3 channels represents the molecular basis of the extraordinary pleiotropy of Andersens syndrome.

Halothane, isoflurane and sevoflurane inhibit NADH:ubiquinone oxidoreductase (complex I) of cardiac mitochondria

We have investigated the effects of volatile anaesthetics on electron transport chain activity in the mammalian heart. Halothane, isoflurane and sevoflurane reversibly increased NADH fluorescence (autofluorescence) in intact ventricular myocytes of guinea‐pig, suggesting that NADH oxidation was impaired. Using pig heart submitochondrial particles we found that the anaesthetics dose‐dependently inhibited NADH oxidation in the order: halothane > isoflurane = sevoflurane. Succinate oxidation was unaffected by either isoflurane or sevoflurane, indicating that these agents selectively inhibit complex I (NADH:ubiquinone oxidoreductase). In addition to inhibiting NADH oxidation, halothane also inhibited succinate oxidation (and succinate dehydrogenase), albeit to a lesser extent. To test the hypothesis that complex I is a target of volatile anaesthetics, we examined the effects of these agents on NADH:ubiquinone oxidoreductase (EC 1.6.99.3) activity using the ubiquinone analogue DBQ (decylubiquinone) as substrate. Halothane, isoflurane and sevoflurane dose‐dependently inhibited NADH:DBQ oxidoreductase activity. Unlike the classical inhibitor rotenone, none of the anaesthetics completely inhibited enzyme activity at high concentration, suggesting that these agents bind weakly to the ‘hydrophobic inhibitory site’ of complex I. In conclusion, halothane, isoflurane and sevoflurane inhibit complex I (NADH:ubiquinone oxidoreductase) of the electron transport chain. At concentrations of ≈2 MAC (minimal alveolar concentration), the activity of NADH:ubiquinone oxidoreductase was reduced by about 20 % in the presence of halothane or isoflurane, and by about 10 % in the presence of sevoflurane. These inhibitory effects are unlikely to compromise cardiac performance at usual clinical concentrations, but may contribute to the mechanism by which volatile anaesthetics induce pharmacological preconditioning.

Comparison of cloned Kir2 channels with native inward rectifier K+ channels from guinea-pig cardiomyocytes.

1 The aim of the study was to compare the properties of cloned Kir2 channels with the properties of native rectifier channels in guinea‐pig (gp) cardiac muscle. The cDNAs of gpKir2.1, gpKir2.2, gpKir2.3 and gpKir2.4 were obtained by screening a cDNA library from guinea‐pig cardiac ventricle. 2 A partial genomic structure of all gpKir2 genes was deduced by comparison of the cDNAs with the nucleotide sequences derived from a guinea‐pig genomic library. 3 The cell‐specific expression of Kir2 channel subunits was studied in isolated cardiomyocytes using a multi‐cell RT‐PCR approach. It was found that gpKir2.1, gpKir2.2 and gpKir2.3, but not gpKir2.4, are expressed in cardiomyocytes. 4 Immunocytochemical analysis with polyclonal antibodies showed that expression of Kir2.4 is restricted to neuronal cells in the heart. 5 After transfection in human embryonic kidney cells (HEK293) the mean single‐channel conductance with symmetrical K+ was found to be 30.6 pS for gpKir2.1, 40.0 pS for gpKir2.2 and 14.2 pS for Kir2.3. 6 Cell‐attached measurements in isolated guinea‐pig cardiomyocytes (n= 351) revealed three populations of inwardly rectifying K+ channels with mean conductances of 34.0, 23.8 and 10.7 pS. 7 Expression of the gpKir2 subunits in Xenopus oocytes showed inwardly rectifying currents. The Ba2+ concentrations required for half‐maximum block at ‐100 mV were 3.24 μm for gpKir2.1, 0.51 μm for gpKir2.2, 10.26 μm for gpKir2.3 and 235 μm for gpKir2.4. 8 Ba2+ block of inward rectifier channels of cardiomyocytes was studied in cell‐attached recordings. The concentration and voltage dependence of Ba2+ block of the large‐conductance inward rectifier channels was virtually identical to that of gpKir2.2 expressed in Xenopus oocytes. 9 Our results suggest that the large‐conductance inward rectifier channels found in guinea‐pig cardiomyocytes (34.0 pS) correspond to gpKir2.2. The intermediate‐conductance (23.8 pS) and low‐conductance (10.7 pS) channels described here may correspond to gpKir2.1 and gpKir2.3, respectively.

Interaction with 14-3-3 proteins promotes functional expression of the potassium channels TASK-1 and TASK-3.

The two‐pore‐domain potassium channels TASK‐1, TASK‐3 and TASK‐5 possess a conserved C‐terminal motif of five amino acids. Truncation of the C‐terminus of TASK‐1 strongly reduced the currents measured after heterologous expression in Xenopus oocytes or HEK293 cells and decreased surface membrane expression of GFP‐tagged channel proteins. Two‐hybrid analysis showed that the C‐terminal domain of TASK‐1, TASK‐3 and TASK‐5, but not TASK‐4, interacts with isoforms of the adapter protein 14‐3‐3. A pentapeptide motif at the extreme C‐terminus of TASK‐1, RRx(S/T)x, was found to be sufficient for weak but significant interaction with 14‐3‐3, whereas the last 40 amino acids of TASK‐1 were required for strong binding. Deletion of a single amino acid at the C‐terminal end of TASK‐1 or TASK‐3 abolished binding of 14‐3‐3 and strongly reduced the macroscopic currents observed in Xenopus oocytes. TASK‐1 mutants that failed to interact with 14‐3‐3 isoforms (V411*, S410A, S410D) also produced only very weak macroscopic currents. In contrast, the mutant TASK‐1 S409A, which interacts with 14‐3‐3‐like wild‐type channels, displayed normal macroscopic currents. Co‐injection of 14‐3‐3ζ cRNA increased TASK‐1 current in Xenopus oocytes by about 70 %. After co‐transfection in HEK293 cells, TASK‐1 and 14‐3‐3ζ (but not TASK‐1ΔC5 and 14‐3‐3ζ) could be co‐immunoprecipitated. Furthermore, TASK‐1 and 14‐3‐3 could be co‐immunoprecipitated in synaptic membrane extracts and postsynaptic density membranes. Our findings suggest that interaction of 14‐3‐3 with TASK‐1 or TASK‐3 may promote the trafficking of the channels to the surface membrane.

Expression Pattern in Brain of TASK-1, TASK-3, and a Tandem Pore Domain K+ Channel Subunit, TASK-5, Associated with the Central Auditory Nervous System

TWIK-related acid-sensitive K(+) (TASK) channels contribute to setting the resting potential of mammalian neurons and have recently been defined as molecular targets for extracellular protons and volatile anesthetics. We have isolated a novel member of this subfamily, hTASK-5, from a human genomic library and mapped it to chromosomal region 20q12-20q13. hTASK-5 did not functionally express in Xenopus oocytes, whereas chimeric TASK-5/TASK-3 constructs containing the region between M1 and M3 of TASK-3 produced K(+) selective currents. To better correlate TASK subunits with native K(+) currents in neurons the precise cellular distribution of all TASK family members was elucidated in rat brain. A comprehensive in situ hybridization analysis revealed that both TASK-1 and TASK-3 transcripts are most strongly expressed in many neurons likely to be cholinergic, serotonergic, or noradrenergic. In contrast, TASK-5 expression is found in olfactory bulb mitral cells and Purkinje cells, but predominantly associated with the central auditory pathway. Thus, TASK-5 K(+) channels, possibly in conjunction with auxiliary proteins, may play a role in the transmission of temporal information in the auditory system.

β-Oxidation of 5-hydroxydecanoate, a Putative Blocker of Mitochondrial ATP-Sensitive Potassium Channels

5‐Hydroxydecanoate (5‐HD) inhibits ischaemic and pharmacological preconditioning of the heart. Since 5‐HD is thought to inhibit specifically the putative mitochondrial ATP‐sensitive K+ (KATP) channel, this channel has been inferred to be a mediator of preconditioning. However, it has recently been shown that 5‐HD is a substrate for acyl‐CoA synthetase, the mitochondrial enzyme which ‘activates’ fatty acids. Here, we tested whether activated 5‐HD, 5‐hydroxydecanoyl‐CoA (5‐HD‐CoA), is a substrate for medium‐chain acyl‐CoA dehydrogenase (MCAD), the committed step of the mitochondrial β‐oxidation pathway. Using a molecular model, we predicted that the hydroxyl group on the acyl tail of 5‐HD‐CoA would not sterically hinder the active site of MCAD. Indeed, we found that 5‐HD‐CoA was a substrate for purified human liver MCAD with a Km of 12.8 ± 0.6 μm and a kcat of 14.1 s−1. For comparison, with decanoyl‐CoA (Km∼3 μm) as substrate, kcat was 6.4 s−1. 5‐HD‐CoA was also a substrate for purified pig kidney MCAD. We next tested whether the reaction product, 5‐hydroxydecenoyl‐CoA (5‐HD‐enoyl‐CoA), was a substrate for enoyl‐CoA hydratase, the second enzyme of the β‐oxidation pathway. Similar to decenoyl‐CoA, purified 5‐HD‐enoyl‐CoA was also a substrate for the hydratase reaction. In conclusion, we have shown that 5‐HD is metabolised at least as far as the third enzyme of the β‐oxidation pathway. Our results open the possibility that β‐oxidation of 5‐HD or metabolic intermediates of 5‐HD may be responsible for the inhibitory effects of 5‐HD on preconditioning of the heart.

Expression pattern and functional characteristics of two novel splice variants of the two-pore-domain potassium channel TREK-2.

Two novel alternatively spliced isoforms of the human two‐pore‐domain potassium channel TREK‐2 were isolated from cDNA libraries of human kidney and fetal brain. The cDNAs of 2438 base pairs (bp) (TREK‐2b) and 2559 bp (TREK‐2c) encode proteins of 508 amino acids each. RT‐PCR showed that TREK‐2b is strongly expressed in kidney (primarily in the proximal tubule) and pancreas, whereas TREK‐2c is abundantly expressed in brain. In situ hybridization revealed a very distinct expression pattern of TREK‐2c in rat brain which partially overlapped with that of TREK‐1. Expression of TREK‐2b and TREK‐2c in human embryonic kidney (HEK) 293 cells showed that their single‐channel characteristics were similar. The slope conductance at negative potentials was 163 ± 5 pS for TREK‐2b and 179 ± 17 pS for TREK‐2c. The mean open and closed times of TREK‐2b at −84 mV were 133 ± 16 and 109 ± 11 μs, respectively. Application of forskolin decreased the whole‐cell current carried by TREK‐2b and TREK‐2c. The sensitivity to forskolin was abolished by mutating a protein kinase A phosphorylation site at position 364 of TREK‐2c (construct S364A). Activation of protein kinase C (PKC) by application of phorbol‐12‐myristate‐13‐acetate (PMA) also reduced whole‐cell current. However, removal of the putative TREK‐2b‐specific PKC phosphorylation site (construct T7A) did not affect inhibition by PMA. Our results suggest that alternative splicing of TREK‐2 contributes to the diversity of two‐pore‐domain K+ channels.

The Retention Factor p11 Confers an Endoplasmic Reticulum-Localization Signal to the Potassium Channel TASK-1

The interaction of the adaptor protein p11, also denoted S100A10, with the C‐terminus of the two‐pore‐domain K+ channel TASK‐1 was studied using yeast two‐hybrid analysis, glutathione S‐transferase pulldown, and co‐immunoprecipitation. We found that p11 interacts with a 40 amino‐acid region in the proximal C‐terminus of the channel. In heterologous expression systems, deletion of the p11‐interacting domain enhanced surface expression of TASK‐1. Attachment of the p11‐interacting domain to the cytosolic tail of the reporter protein CD8 caused retention/retrieval of the construct in the endoplasmic reticulum (ER). Attachment of the last 36 amino acids of p11 to CD8 also caused ER localization, which was abolished by removal or mutation of a putative retention motif (H/K)xKxxx, at the C‐terminal end of p11. Imaging of EGFP‐tagged TASK‐1 channels in COS cells suggested that wild‐type TASK‐1 was largely retained in the ER. Knockdown of p11 with siRNA enhanced trafficking of TASK‐1 to the surface membrane. Our results suggest that binding of p11 to TASK‐1 retards the surface expression of the channel, most likely by virtue of a di‐lysine retention signal at the C‐terminus of p11. Thus, the cytosolic protein p11 may represent a ‘retention factor’ that causes localization of the channel to the ER.

ATP-sensitive potassium channels in capillaries isolated from guinea-pig heart

1 The full‐length cDNAs of two different α‐subunits (Kir6.1 and Kir6.2) and partial cDNAs of three different β‐subunits (SUR1, SUR2A and SUR2B) of ATP‐sensitive potassium (KATP) channels of the guinea‐pig (gp) were obtained by screening a cDNA library from the ventricle of guinea‐pig heart. 2 Cell‐specific reverse‐transcriptase PCR with gene‐specific intron‐spanning primers showed that gpKir6.1, gpKir6.2 and gpSUR2B were expressed in a purified fraction of capillary endothelial cells. In cardiomyocytes, gpKir6.1, gpKir6.2, gpSUR1 and gpSUR2A were detected. 3 Patch‐clamp measurements were carried out in isolated capillary fragments consisting of 3–15 endothelial cells. The membrane capacitance measured in the whole‐cell mode was 19.9 ± 1.0 pF and was independent of the length of the capillary fragment, which suggests that the endothelial cells were not electrically coupled under our experimental conditions. 4 The perforated‐patch technique was used to measure the steady‐state current‐voltage relation of capillary endothelial cells. Application of K+ channel openers (rilmakalim or diazoxide) or metabolic inhibition (250 μm 2,4‐dinitrophenol plus 10 mM deoxyglucose) induced a current that reversed near the calculated K+ equilibrium potential. 5 Rilmakalim (1 μm), diazoxide (300 μm) and metabolic inhibition increased the slope conductance measured at −55 mV by a factor of 9.0 (±1.8), 2.5 (±0.2) and 3.9 (±1.7), respectively. The effects were reversed by glibenclamide (1 μm). 6 Our results suggest that capillary endothelial cells from guinea‐pig heart express KATP channels composed of SUR2B and Kir6.1 and/or Kir6.2 subunits. The hyperpolarization elicited by the opening of KATP channels may lead to an increase in free cytosolic Ca2+, and thus modulate the synthesis of NO and the permeability of the capillary wall.