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.

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. 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. 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. Immunocytochemical analysis with polyclonal antibodies showed that expression of Kir2.4 is restricted to neuronal cells in the heart. 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. 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. 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. 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. 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.

Abstract-ATP-sensitive K (K ATP ) channels are inhibited by cytosolic ATP, a defining property that implicitly links these channels to cellular metabolism. Here we report a direct link between fatty acid metabolism and K ATP channels in cardiac muscle cells. Long-chain (LC) acyl-coenzyme A (CoA) esters are synthesized from fatty acids and serve as the principal metabolic substrates of the heart. We have studied the effects of LC acyl-CoA esters and LC fatty acids on K ATP channels of isolated guinea pig ventricular myocytes and compared them with the effects of phosphatidylinositol 4,5-bisphosphate (PIP 2 ). Application of oleoyl-CoA (0.2 or 1 mol/L), a naturally occurring acyl-CoA ester, to the cytosolic side of excised patches completely prevented rundown of K ATP channels, but not of Kir2 channels. The open probability of K ATP channels measured in the presence of oleoyl-CoA or PIP 2 was voltage dependent, increasing with depolarization. Oleoyl-CoA greatly reduced the ATP sensitivity of K ATP channels. At a concentration of 2 mol/L, oleoyl-CoA increased the half-maximal inhibitory concentration of ATP Ͼ200-fold. The time course of the decrease in ATP sensitivity was much faster during application of oleoyl-CoA than during application of PIP 2 . The effects of PIP 2 , but not of oleoyl-CoA, were inhibited by increasing Ca 2ϩ to 1 mmol/L. Oleate (C18:1; 10 mol/L), the precursor of oleoyl-CoA, inhibited K ATP channels activated by oleoyl-CoA. Palmitoleoyl-CoA and palmitoleate (C16:1) exerted similar reciprocal effects. These findings indicate that LC fatty acids and their CoA-linked derivatives may be key physiological modulators of K ATP channel activity in the heart. (Circ Res. 2001;88:918-924.) Key Words: free fatty acids Ⅲ acyl-CoA esters Ⅲ PIP 2 Ⅲ K ATP channels A TP-sensitive potassium (K ATP ) channels link energy metabolism with the electrical activity of the heart. [1][2][3] This link is functionally important during hypoxia or ischemia. Any imbalance in the ratio between energy supply through coronary arteries and energy expenditure by cardiac myocytes will indirectly modulate electrical activity, because a change in the ATP/ADP ratio, together with acidosis, promotes activation of K ATP channels. 4 -6 By increasing K ϩ efflux and shortening the duration of the action potential, K ATP channel activation reduces transsarcolemmal Ca 2ϩ influx and thereby the energy costs of Ca 2ϩ -ATPases and actomyosin-ATPase. Hence, in the face of reduced energy supply, K ATP channels may provide a means to decrease cytosolic energy demand.We report here a direct effect of long-chain (LC) acylcoenzyme A (CoA) esters and fatty acids on the activity of K ATP channels in isolated cardiac myocytes. We have shown that LC acyl-CoA esters facilitate the opening of K ATP channels by reducing their ATP sensitivity. The precursors of LC acyl-CoA esters, LC fatty acids, were found to inhibit K ATP channels. The reciprocal effects of free fatty acids and acyl-CoA esters on K ATP channels represent a novel link between energy metab...

The potassium channels TASK-1 and TASK-3 show high sequence homology but differ in their sensitivity to extracellular divalent cations. Heterologous expression in HEK293 cells showed that the single-channel conductance of TASK-3 increased approximately four-fold after removal of external divalent cations, whereas the conductance of TASK-1 was unaffected. Replacing the glutamate at position 70 of TASK-3 by a lysine or arginine residue abolished the sensitivity to divalent cations. The reverse mutation in TASK-1 (K70E) induced sensitivity to divalent cations. The organic polycations spermine and ruthenium red modulated the conductance of TASK-3 in a similar way as Ca 2+ or Mg 2+ . Our data suggest that these effects were mediated by shielding of the negative charges in the extracellular loops of TASK-3. Whole-cell currents carried by TASK-3 channels were inhibited by spermine and ruthenium red even in the presence of external divalent cations. These data suggest that, in addition to their effect on single-channel conductance, spermine and ruthenium red decreased the open probability of TASK-3 channels, probably by binding to residue E70. The standing outward current in thalamocortical relay neurons, which is largely carried by TASK channels, was also inhibited by divalent cations and spermine. Using the differential sensitivity of TASK-1 and TASK-3 to divalent cations and spermine we found that about 20% of the standing outward current in thalamocortical relay neurons flows through TASK-3 channels. We conclude from our results that inhibition of TASK-3 channels may contribute to the neuromodulatory effect of spermine released from neurons during repetitive activity or during hypoxia.

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