Methanogenesis and ATP synthesis in methanogenic bacteria at low electrochemical proton potentials. An explanation for the apparent uncoupler insensitivity of ATP synthesis

The rate of methane formation from H2 and CO2, the intracellular ATP content and the electrochemical proton potential (delta mu H+) were determined in cell suspensions of Methanobacterium thermoautotrophicum, which were permeabilized for K+ with valinomycin (1.2 mumol/mg protein). In the absence of extracellular K+ the cells formed methane at a rate of 4 mumol min-1 (mg protein)-1, the intracellular ATP content was 20 nmol/mg protein and the delta mu H+ was 200 mV (inside negative). When K+ was added to the suspensions the measured delta mu H+ decreased to the value calculated from the [K+]in/[K+]out ratio. Using this method of delta mu H+ adjustment, it was found that lowering delta mu H+ from 200 mV ([K+]in/[K+]out = 1000) to 100 mV ([K+]in/[K+]out = 40) had no effect on the rate of methane formation and on the intracellular ATP content. At delta mu H+ values below 100 mV ([K+]in/[K+]out less than 40) both the rate of methanogenesis and the ATP content decreased. Methanogenesis completely ceased and the ATP content was 2 nmol/mg when delta mu H+ was adjusted to values lower 50 mV ([K+]in/[K+]out less than 7). The data show that methanogenesis from H2 and CO2 and ATP synthesis in M. thermoautotrophicum are possible at relatively low electrochemical proton potentials. Similar results were obtained with Methanosarcina barkeri. Protonophoric uncouplers like 3,5,3',4'-tetrachlorosalicylanilide (TCS) or 3,5-di-tert-butyl-4-hydroxy-benzylidenemalononitrile (SF 6847) were found not to dissipate delta mu H+ below 100 mV in M. thermoautotrophicum even when used at high concentrations (400 nmol/mg protein). This finding explains the observed uncoupler insensitivity of methanogenesis and ATP synthesis in this organism.     

The KATP channel Kir6.2 subunit content is higher in glycolytic than oxidative skeletal muscle fibers

It has long been suggested that in skeletal muscle, the ATP-sensitive K(+) channel (K(ATP)) channel is important in protecting energy levels and that abolishing its activity causes fiber damage and severely impairs function. The responses to a lack of K(ATP) channel activity vary between muscles and fibers, with the severity of the impairment being the highest in the most glycolytic muscle fibers. Furthermore, glycolytic muscle fibers are also expected to face metabolic stress more often than oxidative ones. The objective of this study was to determine whether the t-tubular K(ATP) channel content differs between muscles and fiber types. K(ATP) channel content was estimated using a semiquantitative immunofluorescence approach by staining cross sections from soleus, extensor digitorum longus (EDL), and flexor digitorum brevis (FDB) muscles with anti-Kir6.2 antibody. Fiber types were determined using serial cross sections stained with specific antimyosin I, IIA, IIB, and IIX antibodies. Changes in Kir6.2 content were compared with changes in CaV1.1 content, as this Ca(2+) channel is responsible for triggering Ca(2+) release from sarcoplasmic reticulum. The Kir6.2 content was the lowest in the oxidative soleus and the highest in the glycolytic EDL and FDB. At the individual fiber level, the Kir6.2 content within a muscle was in the order of type IIB > IIX > IIA ≥ I. Interestingly, the Kir6.2 content for a given fiber type was significantly different between soleus, EDL, and FDB, and highest in FDB. Correlations of relative fluorescence intensities from the Kir6.2 and CaV1.1 antibodies were significant for all three muscles. However, the variability in content between the three muscles or individual fibers was much greater for Kir6.2 than for CaV1.1. It is suggested that the t-tubular K(ATP) channel content increases as the glycolytic capacity increases and as the oxidative capacity decreases and that the expression of K(ATP) channels may be linked to how often muscles/fibers face metabolic stress.     

Effects of cholesterol on (Na+,K+)-ATPase ATP hydrolyzing activity in bovine kidney

The (Na+,K+)-ATPase ATP hydrolyzing activity from rabbit kidney medulla basolateral membrane vesicles was studied as a function of the cholesterol content of the basolateral membranes. The cholesterol content of the membranes was modified by incubation with phospholipid vesicles. When the cholesterol content was increased above that found in the native membrane, the (Na+,K+)-ATPase ATP hydrolyzing activity was inhibited. When the cholesterol content was decreased from that found in the native membranes, the (Na+,K+)-ATPase ATP hydrolyzing activity was inhibited. Analogous effects were found with the K+-activated phosphatase activity of the same membrane vesicles. Therefore, at low cholesterol contents, cholesterol was stimulatory, and at high cholesterol contents, cholesterol was inhibitory. The structural specificity of this effect was tested by introducing lanosterol and ergosterol as 50% of the membrane sterol. Ergosterol was the least effective at supporting (Na+,K+)-ATPase ATP hydrolyzing activity, while lanosterol was more effective, but still not as effective as cholesterol.     

Character and reasons for change in mitochondrial ATP content during auto-oscillation of ion currents]

The changes in the adenosine triphosphate content in the course of ionic flux oscillations in mitochondria were estimated by using the chemiluminescence method. The ATP concentration changes were shown to be of cyclic character; the oscillations in the ATP content were shifted by 180 degrees C against those of K+ fluxes. The oligomycin-induced oxidative phosphorylation blocking changed (but did not eliminate) the oscillational character of the ATP content in mitochondrial suspensions. It was concluded that substrate phosphorylation is the source of ATP under conditions of oxidative phosphorylation inhibition. Incubation of mitochondria in the presence of exogenous ATP led to suppression of ionic flux oscillations.     

The dependence of intracellular ATP level on the nutrition mode of the acidophilic bacteria Sulfobacillus thermotolerans and Alicyclobacillus tolerans

The dynamics of the ATP pool in the aerobic spore-forming acidothermophilic mixotrophic bacteria Sulfobacillus thermotolerans Kr1T and Alicyclobacillus tolerans K1T were studied in the course of their chemolithoheterotrophic, chemoorganoheterotrophic, and chemolithoautotrophic growth. It was established that, during mixotrophic growth, the maximum ATP concentrations in the cells of S. thermotolerans Kr1 and A. tolerans K1 were 3.8 and 0.6 nmol/mg protein, respectively. The ATP concentrations in sulfobacilli and alicyclobacilli during organotrophic growth were 2.2 and 3.1 nmol/mg protein, respectively. In the cells of the obligately heterotrophic bacterium Alicyclobacillus cycloheptanicus 4006T, the maximum ATP concentration was several times higher and reached 12.3 nmol/mg protein. During lithotrophic growth, the maximum values of the ATP concentration in the cells of S. thermotolerans Kr1 and A. tolerans K1 were 0.3 and <0.1 nmol/mg protein, respectively; in the cells of the autotrophic bacterium Acidithiobacillus ferrooxidans TFBk, the ATP content was about 60-300 times higher (17.0 nmol/mg protein). It is concluded that low ATP content is among the possible causes of growth cessation of S. thermotolerans Kr1 and A. tolerans K1 under auto- and heterotrophic conditions after several culture transfers.     

Effect of membrane potential and internal pH on active sodium-potassium transport and on ATP content in high-potassium sheep erythrocytes.

Ouabain-sensitive Na+ and K+ fluxes and ATP content were determined in high potassium sheep erythrocytes at different values of membrane potential and internal pH. Membrane potential was adjusted by suspending erythrocytes in media containing different concentrations of MgCl2 and sucrose. Concomitantly either the external pH was changed sufficiently to maintain a constant internal pH or the external pH was kept constant with a resultant change of internal pH. The erythrocytes were preincubated before the flux experiment started in a medium which produced increased ATP content in order to avoid substrate limitation of the pump. It was found that an increased cellular pH reduced the rates of active transport of Na+ and K+ without significantly altering the ratio of pumped Na+/K+. This reduction was not due to limitation in the supply of ATP although ATP content decreased when internal pH increased. Changes of membrane potential in the range between -10 and +60 mV at constant internal pH did not affect the rates of active transport of Na+ or K+.     

ATP and integrity of human red blood cells

In spite of the well known significance of ATP in the energy dependent life processes, the role of ATP in maintaining cellular integrity is poorly understood. A possible model for studying ATP dependent life processes is to monitor the kinetics of changes seen intra/extracellularly during ATP depletion. In our model system anticoagulated human whole blood was incubated at different temperatures to reduce intracellular ATP without addition of any chemicals. The red blood cells in their own plasma were incubated for several days at 4 degrees C or at 37 degrees C, and ATP, glucose, K+, Na+, hemoglobin, water content, mean corpuscular volume (MCV), pH and Ca2+ were analyzed in time-sequences. All the examined parameters remained practically unchanged at 4 degrees C, while at 37 degrees C total ATP and glucose decreased parallel and after a transient increase of MCV, the water content of red blood cells decreased. As the actual ATP fell below 10% of the initial ATP content (at 48 h), the release of potassium sharply increased. Release of hemoglobin started only after 96 hours of incubation. Maximums of changes of the examined parameters were found at different time intervals. The maximal speed of concentration changes for glucose was found at 12-24 hours of incubation and at 24-36 hours for ATP, at 48-60 hours for K+(-)Na+ and after 96 hours for hemoglobin.     

The ATP dependence of a ouabain-sensitive sodium efflux activated by external sodium, potassium and lithium in human red cells.

The stimulation of ouabain-sensitive Na+ efflux by external Na+, K+ and Li+ was studied in control and ATP-depleted human red cells. In the presence of 5 mM Na+, with control and depleted cells, Li+ stimulated with a lower apparent affinity than K+, and gave a smaller maximal activation than K+. The ability of Na+, K+ and Li+ to activate Na+ efflux was a function of the ATP content of the cells. Relative to K+ both Na+ and Li+ became more effective activators when the ATP was reduced to about one tenth of the control values. At this low ATP concentration Na+ was absolutely more effective than K+.     

Protein kinase C isoform-dependent modulation of ATP-sensitive K+ channels in mitochondrial inner membrane

The ATP-sensitive K(+) (K(ATP)) channels in both sarcolemmal (sarcK(ATP)) and mitochondrial inner membrane (mitoK(ATP)) are the critical mediators in cellular protection of ischemic preconditioning (IPC). Whereas cardiac sarcK(ATP) contains Kir6.2 and sulfonylurea receptor (SUR)2A, the molecular identity of mitoK(ATP) remains elusive. In the present study, we tested the hypothesis that protein kinase C (PKC) may promote import of Kir6.2-containing K(ATP) into mitochondria. Fluorescence imaging of isolated mitochondria from both rat adult cardiomyocytes and COS-7 cells expressing recombinant Kir6.2/SUR2A showed that Kir6.2-containing K(ATP) channels were localized in mitochondria and this mitochondrial localization was significantly increased by PKC activation with phorbol 12-myristate 13-acetate (PMA). Fluorescence resonance energy transfer microscopy further revealed that a significant number of Kir6.2-containing K(ATP) channels were localized in mitochondrial inner membrane after PKC activation. These results were supported by Western blotting showing that the Kir6.2 protein level in mitochondria from COS-7 cells transfected with Kir6.2/SUR2A was enhanced after PMA treatment and this increase was inhibited by the selective PKC inhibitor chelerythrine. Furthermore, functional analysis indicated that the number of functional K(ATP) channels in mitochondria was significantly increased by PMA, as shown by K(ATP)-dependent decrease in mitochondrial membrane potential in COS-7 cells transfected with Kir6.2/SUR2A but not empty vector. Importantly, PKC-mediated increase in mitochondrial Kir6.2-containing K(ATP) channels was blocked by a selective PKCepsilon inhibitor peptide in both COS-7 cells and cardiomyocytes. We conclude that the K(ATP) channel pore-forming subunit Kir6.2 is indeed localized in mitochondria and that the Kir6.2 content in mitochondria is increased by activation of PKCepsilon. PKC isoform-regulated mitochondrial import of K(ATP) channels may have significant implication in cardioprotection of IPC.     

Use of the luciferin-luciferase assay of ATP for measuring the bacterial growth: Application to Escherichia coli

The relative constancy of ATP content per unit of dry biomass in different microorganisms enables to use the specific, sensitive and quick luciferin-luciferase method for ATP assay for indirect quantitation of microbial biomass by measuring the ATP content in biomass samples.The aim of this work was to investigate the feasibility of using the ATP concentration for the monitoring of the growth of Escherichia coli K 12 W 3350 in different cultivation conditions. Statistically significant high correlation coefficients (r > 0.9) between the ATP content (ATP/ml) and the other growth characteristics examined showed that the ATP content of the culture is as suitable characteristic as the optical density of the culture, total cell number per ml or total cell volume per ml for the monitoring of the growth of E.coliin batch culture in mineral medium with glucose at different growth temperatures (27–42 °C) as well as in fed-batch culture.     

Effects of extracellular ATP on ion transport systems and [Ca2+]i in rat parotid acinar cells. Comparison with the muscarinic agonist carbachol

The effects of extracellular ATP on ion fluxes and the intracellular free Ca2+ concentration ([Ca2+]i) were examined using a suspension of rat parotid acinar cells and were contrasted with the effects of the muscarinic agonist carbachol. Although ATP and carbachol both rapidly increased [Ca2+]i about threefold above the resting level (200-250 nM), the effect of ATP was due primarily to an influx of Ca2+ across the plasma membrane, while the initial response to carbachol was due to a release of Ca2+ from intracellular stores. Within 10 s, ATP (1 mM) and carbachol (20 microM) reduced the cellular Cl- content by 39-50% and cell volume by 15-25%. Both stimuli reduced the cytosolic K+ content by 57-65%, but there were marked differences in the rate and pattern of net K+ movement as well as the effects of K+ channel inhibitors on the effluxes initiated by the two stimuli. The maximum rate of the ATP-stimulated K+ efflux (approximately 2,200 nmol K+/mg protein per min) was about two-thirds that of the carbachol-initiated efflux rate, and was reduced by approximately 30% (vs. 60% for the carbachol-stimulated K+ efflux) by TEA (tetraethylammonium), an inhibitor of the large conductance (BK) K+ channel. Charybdotoxin, another K+ channel blocker, was markedly more effective than TEA on the effects of both agonists, and reduced the rate of K+ efflux initiated by both ATP and carbachol by approximately 80%. The removal of extracellular Ca2+ reduced the ATP- and the carbachol-stimulated rates of K+ efflux by 55 and 17%, respectively. The rate of K+ efflux initiated by either agonist was reduced by 78-95% in cells that were loaded with BAPTA to slow the elevation of [Ca2+]i. These results indicated that ATP and carbachol stimulated the efflux of K+ through multiple types of K(+)-permeable channels, and demonstrated that the relative proportion of efflux through the different pathways was different for the two stimuli. ATP and carbachol also stimulated the rapid entry of Na+ into the parotid cell, and elevated the intracellular Na+ content to 4.4 and 2.6 times the normal level, respectively. The rate of Na+ entry through Na(+)-K(+)-2Cl- cotransport and Na(+)-H+ exchange was similar whether stimulated by ATP, carbachol, or ionomycin, and uptake through these two carrier-mediated transporters accounted for 50% of the ATP-promoted Na+ influx. The remainder may be due to a nonselective cation channel and an ATP-gated cation channel that is also permeable to Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)     

Some factors affecting the response of muscle to insulin

1. The effect of various changes in the composition of the supporting medium on the capacity of isolated rat diaphragm to incorporate amino acids into its protein has been studied. 2. Replacement of most of the normal ionic constituents by sucrose is inhibitory towards protein synthesis, as is also substitution of choline or K(+) for Na(+). 3. The capacity of the tissue to respond to a stimulatory effect of insulin is impaired in the sucrose media and under certain conditions in the absence of Na(+), particularly when Na(+) is replaced by K(+) and the (14)C-labelled amino acid is presented at a relatively high concentration. 4. Cutting of the tissue before incubation also decreases incorporating capacity and markedly decreases responsiveness to insulin. 5. In abnormal media the cellular content of ATP falls sharply. 6. The ATP content of the tissue also declines in the presence of 2-deoxyglucose. This change is prevented by the addition of glucose but not of pyruvate and succinate. 7. Although affecting the rate of amino acid incorporation the ATP content is not thought generally to limit sensitivity to insulin.     

Studies on valinomycin inhibition of synaptosome-fraction protein synthesis.

The ionophore valinomycin inhibited adult and neonatal synaptosome fraction protein synthesis with half-maximal inhibition at approximately 10nM. Valinomycin had no effect on [3H]leucine uptake into synaptosomes at high or low external [K+]. Synaptosome-fraction protein synthesis was dependent on [K+]e reaching a maximum at 25mM-K+. Valinomycin inhibition of protein synthesis was not reversed at high [K+]e. Valinomycin failed to influence the intrasynaptosomal [K+] even at zero [K+]e. A significant increase in State-4 respiration of synaptosomal fractions was found at 5nM-valinomycin with a decrease in the respiratory control index. At these concentrations of valinomycin there was no inhibition of the ADP-stimulated (State 3) respiration rate. Valinomycin had no effect on cerebral microsomal protein synthesis in vitro, which was inhibited by puromycin (100 micrograms/ml) or the absence of ATP. Valinomycin, 2,4-dinitrophenol and KCN inhibition of protein synthesis was not reversed by added ATP, suggesting impermeability of the membrane to ATP. Valinomycin induced a rapid fall in synaptosome ATP content not observed with atractylate or ouabain. Valinomycin inhibition of protein synthesis under these conditions is secondary to uncoupling of mitochondrial oxidative phosphorylation with a subsequent decrease in intraterminal ATP necessary for translation.     

Effects of cellobiose lipid B on Saccharomyces cerevisiae cells: K+ leakage and inhibition of polyphosphate accumulation

Cellobiose lipid B, a natural fungicide produced by the yeast Pseudozyma fusiformata, induces the leakage of K+ and ATP from cells of Saccharomyces cerevisiae. The presence of glucose decreases the effective concentration of cellobiose lipid B. The concentration of cellobiose lipid B was selected that results in a high rate of K+ leakage and a five- to sevenfold decrease in the intracellular ATP content, while the accumulation of acid-soluble polyphosphates decreased only by half. These results indicate the possibility of synthesis of these polymers independently of the ATP level and of the ion gradient on the plasma membrane.     

Two sites for adenine-nucleotide regulation of ATP-sensitive potassium channels in mouse pancreatic β-cells and HIT cells

Summary ATP-inhibited potassium channels (K(ATP)) were studied in excised, inside-out patches from cultured adult mouse pancreatic -cells and HIT cells. In the absence of ATP, ADP opened K(ATP) channels at concentrations as low as 10 m and as high as 500 m, with maximal activation between 10 and 100 m ADP in mouse -cell membrane patches. At concentrations greater than 500 m, ADP inhibited K(ATP) channels while 10 mm virtually abolished channel activity. HIT cell channels had a similar biphasic response to ADP except that more than 1 mm ADP was required for inhibition. The channel opening effect of ADP required magnesium while channel inhibition did not. Using creatine/creatine phosphate solutions with creatine phosphokinase to fix ATP and ADP concentrations, we found substantially different K(ATP)-channel activity with solutions having the same ATP/ADP ratio but different absolute total nucleotide levels. To account for ATP-ADP competition, we propose a new model of channel-nucleotide interactions with two kinds of ADP binding sites regulating the channel. One site specifically binds MgADP and increases channel opening. The other, the previously described ATP site, binds either ATP or ADP and decreases channel opening. This model very closely fits the ADP concentration-response curve and, when incorporated into a model of -cell membrane potential, increasing ADP in the 10 and 100 m range is predicted to compete very effectively with millimolar levels of ATP to hyperpolarize -cells.The results suggest that (i) K(ATP)-channel activity is not well predicted by the ATP/ADP ratio, and (ii) ADP is a plausible regulator of K(ATP) channels even if its free cytoplasmic concentration is in the 10–100 m range as suggested by biochemical studies.We would like to thank Mr. Louis Stamps for expert technical assistance and Dr. Wil Fujimoto and Ms. Jeanette Teague for generously providing HIT cells obtained from Dr. Robert Santerre at Eli Lilly. We would also like to thank Dr. Michel Vivaudou for providing the program ALEX. Support was provided by the NIH and the Department of Veterans Affairs.     

Simultaneous bindings of ATP and vanadate to (Na+ + K+)-ATPase. Implications for the reaction mechanism of the enzyme

Inhibition of (Na+ + K+)-dependent adenosine triphosphatase phosphatase by vanadate is thought to occur through the tight binding of vanadate to the same site from which Pi is released. To see if ATP binds to [48V] vanadate-enzyme complex, just as it does to the phosphoenzyme, the effects of Na+, K+, and ATP on the dissociation rate of the complex at 10 degrees C were studied. The rate constant was increased by Na+, and this increase was blocked by K+, indicating that either Na+ or K+ binds to the complex. ATP alone, or in combination with K+, had no effect on the rate constant. In the presence of Na+, however, ATP caused a further increase in the rate constant. The value of K0.5 of Na+ was the same in the presence or absence of ATP; K0.5 of ATP (0.2 mM) did not seem to change significantly when Na+ concentration was varied, and K0.5 of K+, at a constant Na+ concentration, was the same in the presence or absence of ATP. The data indicate that ATP binds to the enzyme-vanadate complex regardless of the presence or absence of Na+ or K+, but it affects the dissociation rate only when Na+ is bound simultaneously. The value of K0.5 of Na+ decreased as pH was increased in the range of 6.5-7.8, but K0.5 of ATP was independent of pH. Demonstration of ATP binding to the enzyme-vanadate complex provides further support for the suggestion that the oligomeric enzyme contains a low-affinity regulatory site for ATP that is distinct from the interacting high-affinity catalytic sites.     

Direct evidence for ATP consumption due to iodide uptake by isolated, uncultured, pig thyroid epithelial cells

The concept that iodide uptake by thyroid requires a supply of ATP is currently accepted. However, there is little direct evidence that the extents of iodide uptake and ATP consumption are correlated. To demonstrate this correlation, we used isolated, uncultured pig thyroid epithelial cells in basal media containing only glucose and the cations necessary for Na+,K(+)-ATPase, which prevented other possible cellular activities requiring ATP. The isolated cells were sensitive to various metabolic inhibitors of ATP-generating systems, confirming that they were intact. The extents of increase in iodide uptake and decrease in ATP content of the cells were entirely dependent on the presence of the cations, and were closely correlated with each other. Furthermore, the ATP content was not reduced in the absence of NaI, even when all necessary cations for the ATPase were present. These findings provide direct evidence for a correlation between the extents of iodide uptake and ATP consumption. Cells in medium with Mg alone did not show iodide incorporation or ATP consumption, confirming that Mg2(+)-dependent ATPase does not contribute to iodide uptake by the thyroid.     

KATP channel: relation with cell metabolism and role in the cardiovascular system

ATP-sensitive potassium channel (K(ATP)) is one kind of inwardly rectifying channel composed of two kinds of subunits: the pore forming subunits and the regulatory subunits. K(ATP) channels exist in the sarcolemmal, mitochondrial and nuclear membranes of various tissues. Cell metabolism regulates K(ATP) gene expression and metabolism products regulate the channel by direct interactions, while K(ATP) controls membrane potentials and regulate cell activities including energy metabolism, apoptosis and gene expression. K(ATP) channels from different cell organelles are linked by some signal molecules and they can respond to common stimulation in a coordinate way. In the cardiovascular system K(ATP) has important functions. The most prominent is that opening of this channel can protect cardiac myocytes against ischemic injuries. The sarcolemmal K(ATP) may provide a basic protection against ischemia by energy sparing, while both the sarcolemmal K(ATP) and mitochondrial K(ATP) channels are necessary for the ischemia preconditioning. K(ATP) channels also have important functions including homeostasis maintenance and vascular tone regulation under physiological conditions. Further elucidation of the role of K(ATP) in the cardiovascular system will help us to regulate cell metabolism or prevent damage caused by abnormal channel functions.     

Direct activation of cloned K(atp) channels by intracellular acidosis

ATP-sensitive K(+) (K(ATP)) channels may be regulated by protons in addition to ATP, phospholipids, and other nucleotides. Such regulation allows a control of cellular excitability in conditions when pH is low but ATP concentration is normal. However, whether the K(ATP) changes its activity with pH alterations remains uncertain. In this study we showed that the reconstituted K(ATP) was strongly activated during hypercapnia and intracellular acidosis using whole-cell recordings. Further characterizations in excised patches indicated that channel activity increased with a moderate drop in intracellular pH and decreased with strong acidification. The channel activation was produced by a direct action of protons on the Kir6 subunit and relied on a histidine residue that is conserved in all K(ATP). The inhibition appeared to be a result of channel rundown and was not seen in whole-cell recordings. The biphasic response may explain the contradictory pH sensitivity observed in cell-endogenous K(ATP) in excised patches. Site-specific mutations of two residues showed that pH and ATP sensitivities were independent of each other. Thus, these results demonstrate that the proton is a potent activator of the K(ATP). The pH-dependent activation may enable the K(ATP) to control vascular tones, insulin secretion, and neuronal excitability in several pathophysiologic conditions.