Opening of mitochondrial K+ channels increases ischemic ATP levels by preventing hydrolysis

Mitochondrial ATP-sensitive K⁺ channels (mitoK_ATP) have been proposed to mediate protection against ischemic injury by increasing high-energy intermediate levels. This study was designed to verify if mitochondria are an important factor in the loss of cardiac ATP associated to ischemia, and determine the possible role of mitoK_ATP in the control of ischemic ATP loss. Langendorff-perfused rat hearts subjected to ischemia were found to have significantly higher ATP contents when pretreated with oligomycin or atractyloside, indicating that mitochondrial ATP hydrolysis contributes toward ischemic ATP depletion. MitoK_ATP opening induced by diazoxide promoted a similar protection against ATP loss. Diazoxide also inhibited ATP hydrolysis in isolated, nonrespiring mitochondria, an effect accompanied by a drop in the membrane potential and Ca²⁺ uptake. In hearts subjected to ischemia followed by reperfusion, myocardial injury was prevented by diazoxide, but not atractyloside or oligomycin, which, unlike diazoxide, decreased reperfusion ATP levels. Our results suggest that mitoK_ATP-mediated protection occurs due to selective inhibition of mitochondrial ATP hydrolysis during ischemia, without affecting ATP synthesis after reperfusion.     

Small-conductance Ca2+-dependent K+ channels activated by ATP in murine colonic smooth muscle

The patch-clamptechnique was used to determine the ionic conductances activated by ATPin murine colonic smooth muscle cells. Extracellular ATP, UTP, and2-methylthioadenosine 5'-triphosphate (2-MeS-ATP) increasedoutward currents in cells with amphotericin B-perforated patches. ATP(0.5-1 mM) did not affect whole cell currents of cells dialyzedwith solutions containing ethylene glycol-bis(-aminoethylether)-N,N,N',N'-tetraaceticacid. Apamin (3 × 10⁷M) reduced the outward current activated by ATP by 32 ± 5%. Single channel recordings from cell-attached patches showed that ATP, UTP, and2-MeS-ATP increased the open probability of small-conductance, Ca²⁺-dependentK⁺ channels with a slopeconductance of 5.3 ± 0.02 pS. Caffeine (500 µM) enhanced the openprobability of the small-conductance K⁺ channels, and ATP had no effectafter caffeine. Pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid tetrasodium (PPADS,10⁴ M), a nonselectiveP₂ receptor antagonist, preventedthe increase in open probability caused by ATP and 2-MeS-ATP. PPADS hadno effect on the response to caffeine. ATP-induced hyperpolarization inthe murine colon may be mediated byP_2y-induced release of Ca²⁺ from intracellular stores andactivation of the 5.3-pSCa²⁺-activatedK⁺ channels.

     

The electrophysiological expression of Ca2+ channels and of apamin sensitive Ca2+ activated K+ channels is abolished in skeletal muscle cells from mice with muscular dysgenesis

Action potentials of myotubes in culture prepared from 18-19 day -old mouse embryos have a contractile activity and action potentials that are followed by a long lasting after hyperpolarization (ahp) which is blocked by apamin. Myotubes prepared from embryos of mice with muscular dysgenesis (mdg/mdg) did not contract and had action potentials which were never followed by a.h.p.'s. Voltage-clamp experiments have shown that Na+ channel activity was identical in mutant and control muscles and that the activity of fast and slow Ca2+ channels was nearly absent in the mutant.     

Skeletal muscle ATP-sensitive K+ channels recorded from sarcolemmal blebs of split fibers: ATP inhibition is reduced by magnesium and ADP

Summary A new, nonenzymatically treated preparation of amphibian sarcolemmal blebs has been used to study the regulation of skeletal muscle ATP-sensitive K⁺ [K(ATP)] channels.When a frog skeletal muscle fiber is split in half in a Ca²⁺-free relaxing solution, large hemispherical membrane blebs appear spontaneously within minutes without need for Ca²⁺-induced contraction or enzymatic treatment. These blebs readily formed gigaseals with patch pipettes, and excised inside-out patches were found to contain a variety of K⁺ channels. Most prominent were K(ATP) channels similar to those found in the surface membrane of other muscle and nonmuscle cells. These channels were highly selective for K⁺, had a conductance of 53 pS in 140mmK⁺, and were blocked by internal ATP. The presence of these channels in most patches implies that split-fiber blebs are made up, at least in large part, of sarcolemmal membrane.In this preparation, K(ATP) channels could be rapidly and reversibly blocked by glibenclamide (0.1–10 m) in a dose-dependent manner. These channels were sensitive to ATP in the micromolar range in the absence of Mg. This sensitivity was noticeably reduced in the presence of millimolar Mg, most likely because of the ability of Mg²⁺ ions to bind ATP. Our data therefore suggest that free ATP is a much more potent inhibitor of these channels than MgATP. Channel sensitivity to ATP was significantly reduced by ADP in a manner consistent with a competition between ADP, a weak inhibitor, and ATP, a strong inhibitor, for the same inhibitory binding sites.These observations suggest that the mechanisms of nucleotide regulation of skeletal muscle and pancreatic K(ATP) channels are more analogous than previously thought.     

Disopyramide block of K(ATP) channels is mediated by the pore-forming subunit

The class Ia antiarrhythmic agent disopyramide blocks native ATP-sensitive K+ (K(ATP)) channels at micromolar concentrations. The K(ATP) channel is a complex of a pore-forming inwardly rectifying K+ channel (Kir6.2) and a sulfonylurea receptor (SUR). The aim of the present study was to further localize the site of action of disopyramide. We have used a C-terminal truncated form of Kir6.2 (Kir6.2delta26), which--in contrast to Kir6.2--expresses independently of SUR. Kir6.2delta26 channels were expressed in African green monkey kidney COS-7 cells, and enhanced green fluorescent protein (EGFP) cDNA was used as a reporter gene. EGFP fluorescence was visualized by a laser scanning confocal microscope. Disopyramide applied to the cytoplasmic membrane surface of inside-out patches inhibited Kir6.2delta26 channels half-maximally at 7.1 microM (at pH 7.15). Lowering the intracellular pH to 6.5 potentiated the inhibition of Kir6.2delta26 channels by disopyramide. These observations suggest that disopyramide directly blocks the pore-forming Kir6.2 subunit, in particular at reduced intracellular pH values that occur under cardiac ischaemia.     

ATP inhibits pump activity of lymph vessels via adenosine A1 receptor-mediated involvement of NO- and ATP-sensitive K+ channels

We examined the effects of ATP on intrinsic pump activity in lymph vessels isolated from the rat. ATP caused significant dilation with a cessation of lymphatic pump activity. Removal of the endothelium or pretreatment with Nomega-nitro-L-arginine methyl ester (L-NAME) significantly reduced ATP-induced inhibitory responses of lymphatic pump activity, whereas reduction was not suppressed completely by 10(-6) M ATP. L-arginine significantly restored ATP-induced inhibitory responses in the presence of L-NAME. ATP-induced inhibitory responses in lymph vessels with endothelium were also significantly, but not completely, suppressed by pretreatment with glibenclamide. 8-Cyclopentyl-1,3-dipropylxanthine (a selective adenosine A1 receptor antagonist), but not suramine (a P2X and P2Y receptor antagonist) or 3,7-dimethyl-1-proparglyxanthine (a selective adenosine A2 receptor antagonist), significantly decreased ATP-induced inhibitory responses. alpha,beta-methylene ATP (a selective P2X and P2Y receptor agonist) had no significant effect on lymphatic pump activity. In some lymph vessels with endothelium (24 of 30 preparations), adenosine also caused dose-dependent dilation with a cessation of lymphatic pump activity. L-NAME significantly reduced the inhibitory responses induced by the lower (3 x 10(-8)-3 x 10(-7) M) concentrations of adenosine. Glibenclamide or 8-cyclopentyl-1,3-dipropylxanthine also significantly suppressed adenosine-induced inhibitory responses. These findings suggest that ATP-induced dilation and inhibition of pump activity of isolated rat lymph vessels are endothelium-dependent and -independent responses. ATP-mediated inhibitory responses may be, in part, related to production of endogenous nitric oxide, involvement of ATP-sensitive K+ channels, or activation of adenosine A1 receptors in lymphatic smooth muscle and endothelium.     

Activation of inward rectifier K+ channels by hypoxia in rabbit coronary arterial smooth muscle cells

We examined the effects of acute hypoxia on Ba2+-sensitive inward rectifier K+ (K(IR)) current in rabbit coronary arterial smooth muscle cells. The amplitudes of K(IR) current was definitely higher in the cells from small-diameter (<100 microm) coronary arterial smooth muscle cells (SCASMC, -12.8 +/- 1.3 pA/pF at -140 mV) than those in large-diameter coronary arterial smooth muscle cells (>200 microm, LCASMC, -1.5 +/- 0.1 pA pF(-1)). Western blot analysis confirmed that Kir2.1 protein was expressed in SCASMC but not LCASMC. Hypoxia activated much more KIR currents in symmetrical 140 K+. This effect was blocked by the adenylyl cyclase inhibitor SQ-22536 (10 microM) and mimicked by forskolin (10 microM) and dibutyryl-cAMP (500 microM). The production of cAMP in SCASMC increased 5.7-fold after 6 min of hypoxia. Hypoxia-induced increase in KIR currents was abolished by the PKA inhibitors, Rp-8-(4-chlorophenylthio)-cAMPs (10 microM) and KT-5720 (1 microM). The inhibition of G protein with GDPbetaS (1 mM) partially reduced (approximately 50%) the hypoxia-induced increase in KIR currents. In Langendorff-perfused rabbit hearts, hypoxia increased coronary blood flow, an effect that was inhibited by Ba2+. In summary, hypoxia augments the KIR currents in SCASMC via cAMP- and PKA-dependent signaling cascades, which might, at least partly, explain the hypoxia-induced coronary vasodilation.     

Intracellular ADP activates K+ channels that are inhibited by ATP in an insulin-secreting cell line

The effect of ADP on ATP-sensitive K+ channels in the insulin-secreting RINm5F cell line has been investigated with the help of single-channel current recording from saponin-permeabilized cells. ADP (100-500 microM) markedly activates K+ channels when added to the bath solution in contact with the membrane inside. ADP-beta-S cannot mimick this effect. During sustained ATP (500 microM)-evoked inhibition of K+ channel opening, 500 microM ADP markedly and reversibly activates the channels. Conversely ATP markedly reduces the opening probability of ADP-activated channels. It is suggested that the physiological control of K+ channel opening in the insulin-secreting cells is mediated by changes in ATP/ADP ratio rather than being solely determined by the ATP concentration.     

Delayed cardioprotection by isoflurane: role of K(ATP) channels

Isoflurane mimics the cardioprotective effect of acute ischemic preconditioning with an acute memory phase. We determined whether isoflurane can induce delayed cardioprotection, the involvement of ATP-sensitive potassium (K(ATP)) channels, and cellular location of the channels. Neonatal New Zealand White rabbits at 7-10 days of age (n = 5-16/group) were exposed to 1% isoflurane-100% oxygen for 2 h. Hearts exposed 2 h to 100% oxygen served as untreated controls. Twenty-four hours later resistance to myocardial ischemia was determined using an isolated perfused heart model. Isoflurane significantly reduced infarct size/area at risk (means +/- SD) by 50% (10 +/- 5%) versus untreated controls (20 +/- 6%). Isoflurane increased recovery of preischemic left ventricular developed pressure by 28% (69 +/- 4%) versus untreated controls (54 +/- 6%). The mitochondrial K(ATP) channel blocker 5-hydroxydecanoate (5-HD) completely (55 +/- 3%) and the sarcolemmal K(ATP) channel blocker HMR 1098 partially (62 +/- 3%) attenuated the cardioprotective effects of isoflurane. The combination of 5-HD and HMR-1098 completely abolished the cardioprotective effect of isoflurane (56 +/- 5%). We conclude that both mitochondrial and sarcolemmal K(ATP) channels contribute to isoflurane-induced delayed cardioprotection.     

Caffeine-induced impairment of glucose tolerance is abolished by beta-adrenergic receptor blockade in humans.

The caffeine-induced impairment of insulin action is commonly attributed to adenosine receptor (AR) antagonism in skeletal muscle. However, epinephrine, a potent inhibitor of insulin actions, is increased after caffeine ingestion. We tested the hypothesis that the insulin antagonistic effects of caffeine are mediated by epinephrine, and not by AR antagonism, in seven healthy men. On four separate occasions, they received 1) dextrose (placebo, PL), 2) 5 mg/kg caffeine (CAF), 3) 80 mg of propranolol (PR), and 4) 5 mg/kg caffeine + 80 mg of propranolol (CAF + PR) before an oral glucose tolerance test (OGTT). Blood glucose was similar among trials before and during the OGTT. Plasma epinephrine was elevated (P < 0.05) in CAF and CAF + PR. Areas under the insulin and C-peptide curves were 42 and 39% greater (P < 0.05), respectively, in CAF than in PL, PR, and CAF + PR. In the presence of propranolol (CAF + PR), these responses were similar to PL and PR. These data suggest that the insulin antagonistic effects of caffeine in vivo are mediated by elevated epinephrine rather than by peripheral AR antagonism.     

K(ATP) channels mediate the beneficial effects of chronic ethanol ingestion

Chronic ingestion of low doses of ethanol protects the myocardium from ischemic injury by activating adenosine receptors and protein kinase C. We tested the hypothesis that ATP-dependent potassium (K(ATP)) channels mediate these beneficial effects. Dogs were fed with ethanol (1.5 g/kg) or water mixed with dry food twice per day for 12 wk. After they were acutely instrumented for measurement of hemodynamics, dogs received saline (vehicle) or glyburide (0.1 mg/kg iv) and were subjected to 60 min of coronary artery occlusion followed by 3 h of reperfusion. Infarct size (through triphenyltetrazolium chloride staining) was significantly (P < 0.05) reduced to 14 +/- 1% of the left ventricular area at risk in ethanol-pretreated dogs compared with controls (25 +/- 2%). Glyburide alone did not affect infarct size (25 +/- 3%) but abolished the protective effects of ethanol pretreatment (28 +/- 3%). No differences in hemodynamics or coronary collateral blood flow (through radioactive microspheres) were observed among groups. The results indicate that K(ATP) channels mediate the protective effects of chronic consumption of ethanol.     

The ATP-sensitivity of K+ channels in rat pancreatic B-cells is modulated by ADP

ATP-sensitive K+ channels in inside-out membrane patches from dispersed rat pancreatic B-cells were studied using patch-clamp methods. The dose-response curve for ATP-induced channel inhibition was shifted to higher concentrations in the presence of ADP (2 mM). In glucose-free solution, the total intracellular concentration of ATP was 3.8 mM and of ADP 1.5 mM; glucose (20 mM) increased ATP and decreased ADP by approx. 40%. These results suggest that both ADP and ATP may be involved in regulating the activity of the glucose-sensitive K+ channel in intact B-cells.     

ATP consumption by uncoupled mitochondria activates sarcolemmal K(ATP) channels in cardiac myocytes

We tested whether close coupling exists between mitochondria and sarcolemma by monitoring whole cell ATP-sensitive K(+) (K(ATP)) current (I(K,ATP)) as an index of subsarcolemmal energy state during mitochondrial perturbation. In rabbit ventricular myocytes, either pinacidil or the mitochondrial uncoupler dinitrophenol (DNP), which rapidly switches mitochondria from net ATP synthesis to net ATP hydrolysis, had little immediate effect on I(K,ATP). In contrast, in the presence of pinacidil, exposure to 100 microM DNP rapidly activated I(K,ATP) with complex kinetics consisting of a quick rise [time constant of I(K,ATP) increase (tau) = 0.13 +/- 0.01 min], an early partial recovery (tau = 0.43 +/- 0.04 min), and then a more gradual increase. This DNP-induced activation of I(K,ATP) was reversible and accompanied by mitochondrial flavoprotein oxidation. The F(1)F(0)-ATPase inhibitor oligomycin abolished the DNP-induced activation of I(K,ATP). The initial rapid rise in I(K,ATP) was blunted by atractyloside (an adenine nucleotide translocator inhibitor), leaving only a slow increase (tau = 0.66 +/- 0.17 min, P < 0.01). 2,4-Dinitrofluorobenzene (a creatine kinase inhibitor) slowed both the rapid rise (tau = 0.20 +/- 0.01 min, P < 0.05) and the subsequent declining phase (tau = 0.88 +/- 0.19 min, P < 0.05). From single K(ATP) channel recordings, we excluded a direct effect of DNP on K(ATP) channels. Taken together, these results indicate that rapid changes in F(1)F(0)-ATPase function dramatically alter subsarcolemmal energy charge, as reported by pinacidil-primed K(ATP) channel activity, revealing cross-talk between mitochondria and sarcolemma. The effects of mitochondrial ATP hydrolysis on sarcolemmal K(ATP) channels can be rationalized by reversal of F(1)F(0)-ATPase and the facilitation of coupling by the creatine kinase system.     

Saxitoxin is a gating modifier of HERG K+ channels

Potassium (K+) channels mediate numerous electrical events in excitable cells, including cellular membrane potential repolarization. The hERG K+ channel plays an important role in myocardial repolarization, and inhibition of these K+ channels is associated with long QT syndromes that can cause fatal cardiac arrhythmias. In this study, we identify saxitoxin (STX) as a hERG channel modifier and investigate the mechanism using heterologous expression of the recombinant channel in HEK293 cells. In the presence of STX, channels opened slower during strong depolarizations, and they closed much faster upon repolarization, suggesting that toxin-bound channels can still open but are modified, and that STX does not simply block the ion conduction pore. STX decreased hERG K+ currents by stabilizing closed channel states visualized as shifts in the voltage dependence of channel opening to more depolarized membrane potentials. The concentration dependence for steady-state modification as well as the kinetics of onset and recovery indicate that multiple STX molecules bind to the channel. Rapid application of STX revealed an apparent "agonist-like" effect in which K+ currents were transiently increased. The mechanism of this effect was found to be an effect on the channel voltage-inactivation relationship. Because the kinetics of inactivation are rapid relative to activation for this channel, the increase in K+ current appeared quickly and could be subverted by a decrease in K+ currents due to the shift in the voltage-activation relationship at some membrane potentials. The results are consistent with a simple model in which STX binds to the hERG K+ channel at multiple sites and alters the energetics of channel gating by shifting both the voltage-inactivation and voltage-activation processes. The results suggest a novel extracellular mechanism for pharmacological manipulation of this channel through allosteric coupling to channel gating.     

Compromised ATP binding as a mechanism of phosphoinositide modulation of ATP-sensitive K+ channels

Inhibition of ATP-sensitive K(+) (K(ATP)) channels by ATP, a process presumably initiated by binding of ATP to the pore-forming subunit, Kir6.2, is reduced in the presence of phosphoinositides (PPIs). Previous studies led to the hypothesis that PPIs compromise ATP binding. Here, this hypothesis was tested using purified Kir6.2. We show that PPIs bind purified Kir6.2 in an isomer-specific manner, that biotinylated ATP analogs photoaffinity label purified Kir6.2, and that this labeling is weakened in the presence of PPIs. Patch-clamp measurements confirmed that these ATP analogs inhibited Kir6.2 channels, and that PPIs decreased the level of inhibition. These results indicate that interaction of PPIs with Kir6.2 impedes ATP-binding activity. The PPI regulation of ATP binding revealed in this study provides a putative molecular mechanism that is potentially pivotal to the nucleotide sensitivity of K(ATP) channels.     

ATP-sensitive K+ channels in cardiac muscle from cold-acclimated goldfish: characterization and altered response to ATP

ATP-sensitive potassium channels (K(ATP)) play an important, if incompletely defined, role in myocardial function in mammals. With the discovery that K(ATP) channels are also present at high densities in the hearts of vertebrate ectotherms, speculation arises as to their function during periods of cold-acclimation and depressed ATP synthesis. We used single-channel and intracellular recording techniques to examine the possibility that channel activity would be altered in cardiac muscle from goldfish (Carassius auratus) acclimated at 7+/-1 degrees C relative to control (21+/-1 degrees C). As previously observed in mammals, K(ATP) channels in isolated ventricular myocytes were inwardly rectified with slope conductances of 63 pS. However, channel mean open-time and overall open-state probability (Po) were significantly increased in cells from the cold-acclimated animals. In addition, K(ATP) channels in cells from fish acclimated at 7 degrees were nearly insensitive to the inhibitory effects of 2 mM ATP, whether studied at 7 or at 21 degrees C. Transmembrane action potential duration (APD) in hearts of cold-acclimated fish studied at 21 degrees was significantly shorter than that observed in hearts of warm-acclimated fish at the same temperature; this difference was eliminated by the K(ATP) channel antagonist glibenclamide (5 microM). These data suggest that K(ATP) channels in the hearts of cold-acclimated animals are more active and less sensitive to ATP-inhibition than those in warm-acclimated fish, possibly reflecting a functional adaptation to promote tolerance of low temperatures in this species.     

Perinatal hypoxia triggers alterations in K+ channels of adult pulmonary artery smooth muscle cells

Adverse events during the perinatal period, like hypoxia, have been associated with adult diseases. In pulmonary vessels, K(+) channels play an important role in the regulation of vascular tone. In the fetus, Ca(2+)-activated K(+) channels (K(Ca)) are predominant, whereas from birth voltage-gated K(+) channels (K(V)) prevail in the adult. We postulated that perinatal hypoxia could alter this maturational shift and influence regulation of pulmonary vascular tone in relation to K(+) channels in adulthood. We evaluated the effects of perinatal hypoxia on K(V) and K(Ca) channels in the adult main pulmonary artery (PA) using a murine model. Electrophysiological measurements showed a greater outward current in PA smooth muscle cells of mice born in hypoxia than in controls. In controls, only K(V) channels contributed to this current, whereas in mice born in hypoxia both K(V) and K(Ca) channels were implicated. K(V) channel activity was even higher in mice born in hypoxia than in controls. Therefore, perinatal hypoxia results in increased K(Ca) and K(V) channel activity in adult PA. Moreover, PA of adults born in hypoxia displayed higher large-conductance K(Ca) alpha-subunit and K(V)1.5 alpha-subunit protein expression than controls. Interestingly, relaxation induced by nitric oxide (NO) donors [S-nitroso-N-acetyl-D,l-penicillamine, 2-(N,N-diethylamino)-diazenolate-2-oxide] in isolated PA of control mice was not mediated by K(Ca) channels and only slightly by K(V) channels, whereas following perinatal hypoxia both K(Ca) and K(V) channels contributed to this relaxation. Thus perinatal hypoxia results in altered expression and activity of different K(+) channels in the adult main PA, which could contribute to modifications of pulmonary vasoreactivity.