Cytoplasmic ATP Research Articles

GCAC1 recognizes the pH gradient across the plasma membrane: a pH‐sensitive and ATP‐dependent anion channel links guard cell membrane potential to acid and energy metabolism

SummaryIon channels in the plasma membrane of guard cells provide key mechanisms in signal perception and volume regulation during stomatal movement. Recent studies have suggested that the strongly voltage‐dependent, inactivating guard cell anion channel (GCAC1) acts as a sensor of the ambient extracellular CO2 concentration and as a target of modulation by nucleotides and Ca2+ ions. Applying the patch‐clamp technique it is demonstrated here that GCAC1 is activated by cytoplasmic ATP in a pH‐dependent manner.When the apoplastic pH was buffered to 5.6 and the cytosolic pH dropped step‐wise from 7.8 to 5.6, the single‐channel activity increased as a function of proton concentration. This pH‐sensitivity is characterized by a titratable site with an apparent pK value around 6.9. While the steepness and direction of the transmembrane pH gradient did not affect the kinetics of activation, deactivation and fast inactivation of the whole‐cell anion current, the kinetics of slow inactivation and reactivation were strongly influenced. When at a constant intracellular proton concentration of pH 7.2 the external pH decreased from 7.2 to 5.6 the time constants of slow inactivation and the half‐times of reactivation increased two‐ and sevenfold, respectively.The mechanism of nucleotide activation of GCAC1 was quantitatively analysed on the level of single‐channel events. Using inside‐out, cell‐free membrane patches, GCAC1 half‐activated around 0.4 mM ATP. The sigmoidal dose‐dependence of anion channel activation could be well fitted with an apparent Hill coefficient of 3.6. This behaviour might indicate that the activation process involves a strongly cooperative interaction of four ATP‐binding sites. Neither ATP nor its non‐hydrolysable analogue AMP‐PMP, which also activated GCAC1, altered the voltage‐dependent gating. AMP‐PMP stimulation and the insensitivity of GCAC1 towards the phosphatase inhibitor, okadaic acid, and the kinase inhibitors, staurosporine and H‐7, provided evidence that nucleotide binding rather than phosphorylation caused channel activation.Since the time‐ and voltage‐dependent activity of GCAC1 is strongly modulated by ATP and protons, this channel is capable of sensing changes in the energy status, acid metabolism and the H+ ATPase activity of guard cells.

Selective interaction of synthetic antimicrobial peptides derived from sapecin B with lipid bilayers.

By measuring carboxyfluorescein leakage from liposomes and the increase in membrane current through planar lipid bilayer membranes, we examined the capacities of a series of low-molecular-weight cationic amphiphilic peptides derived from the alpha-helix domain of sapecin B for membrane-perturbation and ion-channel formation. Some of these peptides strongly interact with membranes containing acidic phospholipids and phosphatidylethanolamine, with a very negative potential, which are characteristic of the Escherichia coli membrane, in parallel with their antimicrobial activity. In contrast, they do not interact with membranes which predominantly contain choline phospholipids and cholesterol in their outer leaflets, with a slightly negative potential, all of which are characteristic of eukaryotic membranes, thereby providing a molecular basis for their selective toxicity. Membranes doped with these peptides are as permeable to inorganic phosphates as to chloride ions and are far more permeable to cations. The loss of inorganic phosphates may damage bacterial cells due to rapid depletion of cytoplasmic ATP. Examination of the structure-activity relationships of a series of derived peptides in their interaction with a model of the E. coli membrane confirmed the necessity of cationic amphiphilicity for the peptides to attack the bacterial membrane and to exhibit antimicrobial activity.

Evidence for rapid consumption of millimolar concentrations of cytoplasmic ATP during rigor-contracture of metabolically compromised single cardiomyocytes.

Cytoplasmic ATP can be measured continuously in single cardiac myocytes by monitoring the luminescence from microinjected firefly luciferase. We show here that the signals are markedly influenced by changes in cytoplasmic pH, and the calibration of the signals as ATP concentration is markedly affected by cytoplasmic protein. Measurements with a pH-sensitive fluorescent dye show that intracellular pH (pHi) can be clamped at pH 7.08 by perfusing cells with a modified bicarbonate-buffered Krebs saline containing 92 mM NaHCO3 and equilibrated with 20% CO2. Calibration of the firefly luciferase signal in vitro in the presence of high concentrations of BSA (180 mg/ml), to simulate the cytoplasmic protein concentration, revealed a shift in Km (ATP) to 2 mM, from approx. 400 microM in the absence of albumin in an identical ionic milieu. Luciferase measurements in pH-clamped cells show that metabolically poisoned isolated rat ventricle cardiomyocytes enter rigor at a cytoplasmic ATP concentration of between 1 and 2 mM. As the cells shorten in rigor, a process that is complete in 30-40 s, the cytoplasmic ATP concentration falls simultaneously to a level of typically 20 microM. When cyanide is removed 10 min later, to simulate reoxygenation, the signal recovers over a period of 2-3 min to a level approx. 70% of the original in the healthy cell. These studies indicate that rigor-mediated depletion of cytoplasmic ATP in metabolically poisoned cardiomyocytes is considerably more extreme than hitherto indicated.

Protein kinase C-induced changes in the stoichiometry of ATP binding activate cardiac ATP-sensitive K+ channels. A possible mechanistic link to ischemic preconditioning.

Activation of both ATP-sensitive K+ (KATP) channels and the enzyme protein kinase C (PKC) has been associated with the cardioprotective response of ischemic preconditioning. We recently showed that at low cytoplasmic ATP (< or = 50 mumol/L), PKC inhibits KATP channel activity. This finding is surprising, as both KATP channels and PKC are activated during preconditioning. However, PKC also altered ATP binding to the channel, changing the Hill coefficient from approximately 2 to approximately 1. This apparent change in stoichiometry would lead to a PKC-induced activation of KATP channels at more physiological (millimolar) levels of ATP. The aim of the present study was to determine whether PKC activates cardiac KATP channels at millimolar levels of ATP. The effects of PKC on single KATP channels were studied at millimolar internal ATP levels using excised inside-out membrane patches from rabbit ventricular myocytes. Application of purified constitutively active PKC (20 nmol/L) to the intracellular surface of the patches produced an approximately threefold increase in the channel open probability. The specific PKC inhibitor peptide PKC(19-31) prevented this increase. Heat-inactivated PKC had no effect on KATP channel properties. KATP channel activity spontaneously returned to control levels after washout of PKC. This spontaneous reversal did not occur in the presence of 5 nmol/L okadaic acid, suggesting that the reversal of PKC's action is dependent on activity of a membrane-associated type 2A protein phosphatase (PP2A). In the presence of exogenous PP2A (7.5 nmol/L), PKC had no effect. We conclude that the PKC-induced increase in KATP channel activity at millimolar ATP results from a crossing of the ATP concentration-response curves for inhibition of the phosphorylated and nonphosphorylated forms of the channel. This identifies a mechanism by which PKC activates KATP channels at near physiological levels of ATP and thus could link these two components in a signaling pathway that induces ischemic preconditioning.

Glycolytic Flux and Hexokinase Activities in Anoxic Maize Root Tips Acclimated by Hypoxic Pretreatment.

Several enzyme activities were measured in extracts from acclimated and nonacclimated maize (Zea mays) root tips at pH 6.5 and 7.5, corresponding to cytoplasmic pH in anaerobiosis or aerobiosis, respectively, to determine what causes the decline of the glycolytic flux observed in anoxia in nonacclimated tips. We found that phosphorylation of hexoses by kinases was a major limiting step of glycolysis in anoxia. When fructose was substituted for glucose, glycolysis was slightly enhanced and survival improved, but neither matched that of acclimated tips. Decrease of kinase activities was not the result of proteolytic degradation but was more likely the result of inhibition by internal factors (low pH and low ATP). There was no evidence of induction during the hypoxic pretreatment of isoenzymes better adapted to the anoxic cellular environment. Maintenance of the glycolytic flux in acclimated tissues is explained by a combination of a rise in kinase activities and decreased inhibition resulting from a higher cytoplasmic pH and ATP content. The behavior of intact root tips is discussed in comparison with the behavior of excised root tips.

A strongly inwardly rectifying K+ channel that is sensitive to ATP

We have cloned an inwardly rectifying K+ channel from the hamster insulinoma cDNA library and shown that it is inhibited by cytoplasmic ATP. The channel is 90.97% identical to the IRK3 channels cloned from other species, and its mRNA is found primarily in the brain. When expressed in Xenopus oocytes, the channel displays strong inward rectification typical of inward rectifiers. The channel is inhibited reversibly by physiological concentrations of ATP via a mechanism that does not appear to involve ATP hydrolysis, as shown by studies of channels in excised inside-out membrane patches. This effect is antagonized by ADP, again in the physiological range, implying that this channel is sensitive to the index of metabolic state, i.e., the intracellular [ATP]/[ADP] ratio. This channel is different from previously known ATP-sensitive K+ channels, although it may also be stimulated by MgATP, as are other ATP-sensitive K+ channels. The potential physiological significance of these ATP-dependent regulations will be discussed.

Identification and partial purification of PAPS translocase.

Sulfation of all macromolecules in higher organisms requires the high-energy donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS). PAPS is synthesized via the sequential actions of two cytoplasmic enzymes, ATP sulfurylase and APS kinase, and then must be transferred across the Golgi membrane for utilization by lumenal sulfotransferases. Following the kinetic characterization of the PAPS translocase as a specific transporter that act through an antiport mechanism with PAP as the returning ligand [Ozeran, J.D., Westley, J., Schwartz, N.B. (1996) Biochemistry 35, 3685-3694 (accompanying paper)], the present study describes the identification and physical characterization of the PAPS translocase from rat liver Golgi membranes. The following evidence suggests the PAPS translocase is a membrane spanning protein of approximately 230 kDa: isolation by affinity chromatography on beta-methylene PAPS matrices of a 230 kDa Golgi membrane protein concomitant with PAPS translocase activity; demonstration that the 230 kDa protein possesses the only PAPS binding site accessible to the cytoplasmic face of intact Golgi membranes, while several other PAPS binding proteins are labeled in solubilized membrane preparations; reduction in size of the 230 kDa membrane protein and loss of PAPS translocase activity following protease treatment; estimation via hydrodynamic analysis of a molecular size of the membrane protein associated with PAPS translocase activity; and correlation of beta-methylene PAPS binding and labeling of the 230 kDa Golgi protein with PAPS translocase activity in artificial liposomes. These and the accompanying data have permitted the identification of the first of a potentially large class of Golgi membrane nucleotide-metabolite transporters.

Hyperammonemia inhibits platelet aggregation in rats

The effect of hyperammonemia on ex vivo platelet function and in vivo nitric oxide synthesis was evaluated in rats. In addition, mitochondrial energy production was assessed from the fluorescence intensity of tetramethylrhodamine ethyl ester (TMRE). Continuous ammonium acetate infusion significantly reduced ex vivo platelet aggregation concomitant with a decrease of the platelet cytoplasmic ATP level. The serum level of L-arginine, as well as the levels of nitrite and nitrate (oxidative by-products of nitric oxide), increased with ammonium infusion. Prior administration of N ω-nitro-L-arginine methyl ester, a competitive inhibitor of nitric oxide synthase, did not affect the ammonia-induced rise in L-arginine, but substantially attenuated the associated decrease of platelet ATP and TMRE fluorescence as well as diminishing the anti-aggregatory effect of ammonia infusion. These findings suggest that the synthesis of nitric oxide from L-arginine is accelerated by continuous ammonium infusion and inhibits ex vivo platelet aggregation in the rat, probably by reducing mitochondrial energy production.

Molecular biological and quantitative abnormalities of ADP/ATP carrier protein in cardiomyopathic hamsters.

The adenine nucleotide translocator or ADP/ATP carrier protein (AAC) is an integral protein present in the inner mitochondrial membrane, which performs the exchange of cytoplasmic and intramitochondrial ADP and ATP. The myocardial AAC content was studied in J-2-N cardiomyopathic hamsters. The AAC content was found to be significantly decreased in J-2-N hamsters. For molecular biological analysis, hamster AAC (T1 isoenzyme) cDNA was cloned by the plaque hybridization method. This AAC cDNA hybridized specifically with AAC mRNA, so RNA dot-blot hybridization was performed. The highest AAC mRNA level was observed in control hamsters followed by J-2-N hamsters with mild myocardial damage, J-2-N hamsters with severe myocardial damage and Bio 14-6 cardiomyopathic hamsters. These results suggest that a decreased AAC content may contribute to the pathogenesis of cardiomyopathy and that a decrease of AAC mRNA levels may explain the abnormalities of AAC in J-2-N cardiomyopathic hamsters.

Modulation of Na +,Ca 2+ exchange current by EGTA calcium buffering in giant cardiac membrane patches

Effects of calcium buffering by EGTA were examined on sodium-calcium exchange currents ( I Naca) in inside-out giant cardiac membrane patches. Free calcium concentrations (Ca 2+) were monitored with a calcium electrode and a fluorescent calcium indicator (Calcium Green-5N). With 1.8 μM cytoplasmic Ca 2+, inward I Naca increased 2-fold at −120 mV when EGTA concentration was increased from 0.1 mM to 10 mM (37°C and 140 mM extracellular sodium). Stimulation by EGTA was decreased or abolished under conditions of attenuated exchanger turnover rate: temperature < 30°C, extracellular sodium < 70 mM, and membrane potential > + 60 mV. EGTA concentration had no effect on outward I Naca with 100 mM cytoplasmic Na + and 0.8 μM cytoplasmic Ca 2+, conditions under which the current inactivated by about 70%. EGTA (0.1–10 mM) and BAPTA (10 mM) inhibited the current by about 80% when the outward I Naca was stimulated by 2 mM cytoplasmic ATP or by phosphatidylserine. The apparent K i for EGTA was 0.2 mM. The electroneutral calcium ionophore, A23187, activated outward I Naca even in presence of 10 mM EGTA. Our results are consistent with EGTA acting as a simple calcium buffer with no direct effect on the exchanger. At low concentrations of EGTA, inhibition of the inward I Naca is expected due to submembrane calcium depletion by the exchanger; enhancement of the outward I Naca at low EGTA concentrations is expected because submembrane calcium accumulates and activates I Naca via regulatory calcium binding sites.

Efflux of ions and ATP depletion induced by pediocin PA‐1 are concomitant with cell death in Listeria monocytogenes Scott A

The bacteriocin pediocin PA‐1 (80 AU ml−1), which is produced by Pediococcus acidilactici PAC 1.0, reduced Listeria monocytogenes Scott A viability by 4.6 log. Concomitant with the cell death, pediocin PA‐1 induced irreversible K+ and Pi efflux and ATP depletion in concentration‐dependent and time‐dependent fashions. The maximum amount of intracellular K+ and Pi loss was 520 and 44.6 nmol mg−1 CDW, respectively. Intracellular ATP levels decreased from 2.8 to 0‐029 nmol mg−1 CDW, corresponding to 98.9% depletion. Less than 0.1% ATP efflux was found with pediocin concentrations up to 200 AU ml−1. Approximately 10, 40 and 60 min were required for maximum K+ efflux, ATP depletion and Pi efflux, respectively. Moreover, pediocin PA‐1 depleted 90% of cytoplasmic ATP when only 25% Pi efflux took place. These results suggest that loss of ATP is due to attempts by the cell to maintain proton‐motive force rather than the inability of the cell to produce ATP due to the loss of Pi. Dithiothreitol‐treated PA‐1 no longer inhibited the growth of L. monocytogenes cells, and caused neither concentration‐dependent ion efflux nor ATP depletion.

Membrane transport mechanisms probed by capacitance measurements with megahertz voltage clamp.

We have used capacitance measurements with a 1-microsecond voltage clamp technique to probe electrogenic ion-transporter interactions in giant excised membrane patches. The hydrophobic ion dipicrylamine was used to test model predictions for a simple charge-moving reaction. The voltage and frequency dependencies of the apparent dipicrylamine-induced capacitance, monitored by 1-mV sinusoidal perturbations, correspond to single charges moving across 76% of the membrane field at a rate of 9500 s-1 at 0 mV. For the cardiac Na,K pump, the combined presence of cytoplasmic ATP and sodium induces an increase of apparent membrane capacitance which requires the presence of extracellular sodium. The dependencies of capacitance changes on frequency, voltage, ATP, and sodium verify that phosphorylation enables a slow, 300- to 900-s-1, pump transition (the E1-E2 conformational change), which in turn enables fast, electrogenic, extracellular sodium binding reactions. For the GAT1 (gamma-aminobutyric acid,Na,Cl) cotransporter, expressed in Xenopus oocyte membrane, we find that chloride binding from the cytoplasmic side, and probably sodium binding from the extracellular side, results in a decrease of membrane capacitance monitored with 1- to 50-kHz perturbation frequencies. Evidently, ion binding by the GAT1 transporter suppresses an intrinsic fast charge movement which may originate from a mobility of charged residues of the transporter binding sites. The results demonstrate that fast capacitance measurements can provide new insight into electrogenic processes closely associated with ion binding by membrane transporters.

Molecular characterization of an Arabidopsis thaliana cDNA encoding a novel putative adenylate translocator of higher plants

We have isolated an Arabidopsis thaliana cDNA encoding a highly hydrophobic membrane protein of 589 amino acids which contains 12 potential transmembrane helices and shows a high degree of similarity (43.5% identity, 66.2% similarity) to the ATP/ADP translocase of the Gram-negative bacterium Rickettsia prowazekii, an obligate intracellular parasite responsible for the epidemic typhus. This rickettsial translocator resides in the cytoplasmic membrane and allows the bacterium to exploit the host cytoplasmic ATP pool. We hypothesize that the A. haliana homolog of the R. prowazekii ATP/ADP translocase is the functional eukaryotic equivalent and resides in the plastid inner envelope membrane where it functions as an ATP importer.

Creatine kinase is required for swelling-activated K-Cl cotransport in dog red blood cells.

K-Cl cotransport in resealed dog red cell ghosts requires the incorporation of creatine phosphate before resealing; incorporation of ATP has no effect [Colclasure and Parker. Am. J. Physiol. 265 (Cell Physiol. 34): C1648-C1652, 1993]. A role for creatine kinase (CK) in swelling-activated K-Cl cotransport was investigated. 2,4-Dinitrofluorobenzene (DNFB), an inhibitor of CK, inhibited K-Cl cotransport in intact red blood cells and resealed ghosts from DNFB-treated cells. Incorporation of exogenous CK into ghosts of DNFB-treated cells restored K-Cl cotransport. Therefore DNFB inhibits CK and not the cotransporter. Inhibition of native CK in ghosts by DNFB and the incorporation of CK into the ghosts were demonstrated in electrophoretic gels. In a dose-response experiment, approximately 770 molecules CK/ghost restored 50% of control cotransport. Since creatine phosphate is a substrate only for CK, CK provides ATP to a site inaccessible to cytoplasmic ATP. The nature of this site and its role in K-Cl cotransport are uncertain, but an essential function for CK is established.

Mechanism of high-affinity potassium uptake in roots of Arabidopsis thaliana.

Potassium is a major nutrient in higher plants, where it plays a role in turgor regulation, charge balance, leaf movement, and protein synthesis. Terrestrial plants are able to sustain growth at micromolar external K+ concentrations, at which K+ uptake across the plasma membrane of root cells must be energized despite the presence of a highly negative membrane potential. However, the mechanism of energization has long remained obscure. Therefore, whole-cell mode patch clamping has been applied to root protoplasts from Arabidopsis thaliana to characterize membrane currents resulting from the application of micromolar K+. Analysis of whole cell current/voltage relationships in the presence and absence of micromolar K+ enabled direct testing of K+ transport for possible energization by cytoplasmic ATP and the respective trans-membrane gradients of Na+, Ca2+, and H+. Subtracted current/voltage relations for K(+)-dependent membrane currents are independent of ATP and reverse at potentials that imply H(+)-coupled K+ transport with a ratio of 1 H+:K+. Furthermore, the reversal potential of the K+ current shifts negative as external H+ activity is decreased. K(+)-dependent currents saturate in the micromolar concentration range with an apparent Km of 30 microM, a value in close agreement with previously reported Km values for high-affinity K+ uptake. We conclude that our results are consistent with the view that high-affinity K+ uptake in higher plants is mediated by a H+:K+ symport mechanism, competent in driving K+ accumulation to equilibrium ratios in excess of 10(6)-fold.

Sodium transport through the amiloride-sensitive Na-Mg pathway of hamster red cells

Previous work showed that in hamster red cells the amiloride-sensitive (AS) Na+ influx of 0.8 mmol/liter cells/hr is not mediated by Na-H exchange as in other red cells, but depends upon intracellular Mg2+ and can be increased by 40-fold by loading cells with Mg2+ to 10 mM. The purpose of this study was to verify the connection of AS Na+ influx with Na-dependent, amiloride-sensitive Mg2+ efflux and to utilize AS Na+ influx to explore that pathway. Determination of unidirectional influx of Na+ and net loss of Mg2+ in parallel sets of cells showed that activation by extracellular [Na+] follows a simple Michaelis-Menten relationship for both processes with a Km of 105-107 mM and that activation of both processes is sigmoidally dependent upon cytoplasmic [Mg2+] with a [Mg2+]0.5 of 2.1-2.3 mM and a Hill coefficient of 1.8. Comparison of Vmax for both sets of experiments indicated a stoichiometry of 2 Na:1 Mg. Amiloride inhibits Na+ influx and Mg2+ extrusion in parallel (Ki = 0.3 mM). Like Mg2+ extrusion, amiloride-sensitive Na+ influx shows an absolute requirement for cytoplasmic ATP and is increased by cell swelling. Hence, amiloride-sensitive Na+ influx in hamster red cells appears to be through the Na-Mg exchange pathway. There was no amiloride-sensitive Na+ efflux in hamster red cells loaded with Na+ and incubated with high [Mg2+] in the medium with or without external Na+, nor with ATP depletion. Hence, this is not a simple Na-Mg exchange carrier.

The Influence of Hypoxia and Anoxia on Distribution of Adenine Nucleotides in Isolated Hepatocytes

Isolated hepatocytes incubated under conditions of "chemical hypoxia" (KCN + iodoacetic acid) exhibited a marked dephosphorylation of the cytoplasmic and mitochondrial adenine nucleotides to AMP. Cytoplasmic adenine nucleotide levels (ATP + ADP + AMP) were decreased by 40%. There was no significant change in the mitochondrial adenine nucleotide pool size. For starved rats, but not for fed rats, addition of KCN to isolated hepatocytes resulted in a shift of the mitochondrial adenine nucleotide species to AMP. This difference was correlated with the maintenance of a substantial level of cytoplasmic ATP in the fed vs starved condition. The addition of fructose (but not glucose) to hepatocytes isolated from a starved rat, prevented the KCN-induced dephosphorylation of mitochondrial adenine nucleotides to AMP. Fructose-treated cells had a significant level of ATP in the cytoplasm, whereas glucose-treated cells did not. Addition of A23187 to fructose-treated (but not glucose-treated) cells resulted in a net loss in the mitochondrial adenine nucleotide content. The results suggest that the shift of matrix adenine nucleotides from ATP and ADP to AMP preserves the mitochondrial adenine nucleotide pool size during transient hypoxia by preventing net adenine nucleotide transport to the cytoplasm via the ATP-Mg/Pi carrier. This effectively protects those adenine nucleotides from the cytoplasmic purine degradation pathway, a strategy that has the potential to facilitate rapid recovery of bioenergetic status by oxidative phosphorylation upon reoxygenation.

Effect of cAMP on intracellular and extracellular ATP content of Cl- -secreting epithelia and 3T3 fibroblasts.

ATP may play an important role in the regulation of Cl- secretion in epithelial cells. Previous studies have suggested that ATP may regulate Cl- secretion in at least two ways. First, cytoplasmic ATP is required for the opening of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels. Thus, an increase in metabolic activity associated with cAMP-stimulated Cl-secretion might decrease cellular levels of ATP, thereby decreasing the activity of CFTR Cl- channels. Second, cAMP-dependent opening of CFTR channels might allow the efflux of ATP through the channels into the mucosal solution. An increase in extracellular ATP could in turn activate apical membrane receptors that stimulate other Cl- channels. We tested the hypothesis that acute stimulation of Cl- secretion would produce changes in cell ATP concentration and/or in ATP concentration in the mucosal fluid. We found that in an intestinal epithelial cell line (T84) and in primary cultures of airway epithelia, cAMP-dependent stimulation of Cl- secretion did not alter cell ATP content. Moreover, cAMP agonists did not increase the ATP or nucleotide content of the mucosal fluid. In contrast, when 3T3 fibroblasts stably expressing CFTR were stimulated with cAMP agonists, cell ATP levels decreased. In addition, 3T3 cells appear to have a pathway that allows leakage of ATP into the extracellular fluid; this pathway is not affected by the presence or activation of CFTR. We interpret the data to suggest that changes in intracellular ATP probably do not regulate CFTR in Cl- -secreting epithelia under physiologic conditions and that secretion of ATP probably plays a minor role in control of transepithelial Cl-transport.

Inhibition of pinacidil induced IK(ATP) in heart by changes in extracellular pH.

The aim was to determine whether extracellular protons influence the activation of ATP sensitive potassium channels (KATP) in heart by the drug pinacidil. Single channel and whole cell currents were measured in enzymatically dispersed guinea pig ventricular myocytes with patch clamp procedures. ATP sensitive potassium channel activity was induced by pinacidil in whole cell experiments and either by lowering cytoplasmic ATP or pinacidil in membrane patch experiments. Extracellular acidification inhibited and extracellular alkalisation potentiated ATP sensitive potassium channel currents (IK(ATP)) induced by pinacidil under whole cell conditions. In membrane patches, IK(ATP) induced by low cytoplasmic ATP (1 mM) was not sensitive to changes in extracellular pH, but IK(ATP) induced by cytoplasmic application of pinacidil was inhibited by extracellular acidification. Interactions between protons and pinacidil drug molecules underlie the pH dependent changes in IK(ATP) induced by pinacidil. Based on the kinetics of pH dependent changes in pinacidil induced channel activity in inside-out membrane patches, our data suggest that the important inhibitory site of action of this drug is located on the extracellular side of the cell membrane.

KATP channels in vascular smooth muscle

ATP sensitive potassium channels (KATP channels) appear widely distributed in the vascular system. At the single channel level, channels with both small and large conductance have been described, though the former appear to be activated by potassium channel openers or ATP depletion in whole cell studies. KATP channels are inhibited by cytoplasmic ATP, and may be activated by intracellular nucleotide diphosphates. Regulation by intracellular metabolites confers a degree of sensitivity of the channel to the metabolic status of the cell, and there is evidence that KATP currents are activated during metabolic inhibition. In general, activation of KATP channels will lead to membrane hyperpolarisation and so to vasorelaxation. The functional role of the channel is being intensively studied at present. The channel may form a target for a number of endogenous vasodilators, and may be inhibited by some vasoconstrictors. It may be involved in the vasodilator response to hypoxia, and may contribute to the resting membrane potential of smooth muscle in some blood vessels.