Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Transepithelial Cl- secretion in vertebrates is accomplished by a secondary active transport process brought about by the coordinated activity of apical and basolateral transport proteins. The principal basolateral components are the Na+/K(+)-ATPase pump, the Na+/K+/2Cl- cotransporter (symporter) and a K+ channel. The rate-limiting apical component is a cyclic-AMP-stimulated Cl- channel. As postulated nearly two decades ago, the net Cl- movement from the blood to the lumen involves entry into the epithelial cells with Na+ and K+, followed by active Na+ extrusion via the pump and passive K+ exit via a channel. Intracellular [Cl-] is raised above electrochemical equilibrium and exits into the lumen when the apical Cl- channel opens. Cl- secretion is accompanied by a passive paracellular flow of Na+. The tubules of the rectal glands of elasmobranchs are highly specialized for secreting concentrated NaCl by this mechanism and hence have served as an excellent experimental model in which to characterize the individual steps by electrophysiological and ion flux measurements. The recent molecular cloning and heterologous expression of the apical Cl- channel and basolateral cotransporter have enabled more detailed analyses of the mechanisms and their regulation. Not surprisingly, since hormones acting through kinases control secretion, both the Cl- channel, which is the shark counterpart of the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator), and the cotransporter are regulated by phosphorylation and dephosphorylation. The primary stimulation of secretion by hormones employing cyclic AMP as second messenger activates CFTR via the direct action of protein kinase A (PKA), which phosphorylates multiple sites on the R domain. In contrast, phosphorylation of the cotransporter by as yet unidentified kinases is apparently secondary to the decrease in intracellular chloride concentration caused by anion exit through CFTR.
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PMID:The molecular basis of chloride transport in shark rectal gland. 752 18

CFTR is a member of the traffic ATPase superfamily and a Cl- ion channel that appears to require ATP hydrolysis for gating. Analysis of single CFTR Cl- channels reconstituted into planar lipid bilayers revealed the presence of two open conductance states that are connected to each other and to the closed state by an asymmetric cycle of gating events. We show here that the transition between the two open conductance states is directly coupled to ATP hydrolysis by one of the consensus nucleotide-binding folds, designated NBF2. Moreover, the transition between the closed state and one of the open states is linked to the binding of ATP. This analysis permits real-time visualization of conformational changes associated with a single cycle of ATP hydrolysis by a single protein molecule and suggests a model describing a role for ATP in CFTR gating.
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PMID:Conformational states of CFTR associated with channel gating: the role ATP binding and hydrolysis. 754 23

Cystic fibrosis is caused by mutations in the cell membrane protein called CFTR (cystic fibrosis transmembrane conductance regulator) which functions as a regulated Cl- channel. Although it is known that CFTR contains two nucleotide domains, both of which exhibit the capacity to bind ATP, it has not been demonstrated directly whether one or both domains can function as an active ATPase. To address this question, we have studied the first CFTR nucleotide binding fold (NBF1) in fusion with the maltose-binding protein (MBP), which both stabilizes NBF1 and enhances its solubility. Three different ATPase assays conducted on MBP-NBF1 clearly demonstrate its capacity to catalyze the hydrolysis of ATP. Significantly, the mutations K464H and K464L in the Walker A consensus motif of NBF1 markedly impair its catalytic capacity. MBP alone exhibits no ATPase activity and MBP-NBF1 fails to catalyze the release of phosphate from AMP or ADP. The Vmax of ATP hydrolysis (approximately 30 nmol/min/mg of protein) is significant and is markedly inhibited by azide and by the ATP analogs 2'-(3')-O-(2,4,6-trinitrophenyl)-adenosine-5'-triphosphate and adenosine 5'-(beta, gamma-imido)triphosphate. As inherited mutations within NBF1 account for most cases of cystic fibrosis, results reported here are fundamental to our understanding of the molecular basis of the disease.
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PMID:The first nucleotide binding fold of the cystic fibrosis transmembrane conductance regulator can function as an active ATPase. 754 72

These results begin to indicate that nucleoside triphosphates directly regulate CFTR Cl- channels by interacting with the NBDs. Thus, they may begin to explain why some CF-associated mutations in the NBDs may block Cl- channel function in the epithelia of CF patients. These results also suggest that the intracellular ATP/ADP ratio may be more important than the absolute concentration of ATP in regulating CFTR. Thus, changes in the metabolic state of the cell that alter the ATP-ADP ratio may regulate CFTR Cl- channel activity in vivo. These observations suggest that CFTR might be regulated in the physiologic range of nucleotides. Such a mechanism of regulation could provide a mechanism for coupling the metabolic status of the cell and the activity of the Na-K ATPase with the rate of transepithelial Cl- secretion as regulated by apical membrane CFTR Cl- channels.
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PMID:Regulation of the cystic fibrosis transmembrane conductance regulator chloride channel by MgATP. 768 67

The maltose transport system of Escherichia coli is a well-characterized member of the ATP binding cassette transporter superfamily. Members of this family share sequence similarity surrounding two short sequences (the Walker A and B sequences) which constitute a nucleotide binding pocket. It is likely that the energy from binding and hydrolysis of ATP is used to accomplish the translocation of substrate from one location to another. Periplasmic binding protein-dependent transport systems, like the maltose transport system of E.coli, possess a water-soluble ligand binding protein that is essential for transport activity. In addition to delivering ligand to the membrane-bound components of the system on the external face of the membrane, the interaction of the binding protein with the membrane complex initiates a signal that is transmitted to the ATP binding subunit on the cytosolic side and stimulates its hydrolytic activity. Mutations that alter the membrane complex so that it transports independently of the periplasmic binding protein also result in constitutive activation of the ATPase. Genetic analysis indicates that, in general, two mutations are required for binding protein-independent transport and constitutive ATPase. The mutations alter residues that cluster to specific regions within the membrane spanning segments of the integral membrane components MalF and MalG. Individually, the mutations perturb the ability of MBP to interact productively with the membrane complex. Genetic alteration of this signalling pathway suggests that other agents might have similar effects. These could be potentially useful for modulating the activities of ABC transporters such as P-glycoprotein or CFTR, that are implicated in disease.
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PMID:Mutations that alter the transmembrane signalling pathway in an ATP binding cassette (ABC) transporter. 815 12

CFTR shares structural homology with the ABC transporter superfamily of proteins which hydrolyze ATP to effect the transport of compounds across cell membranes. Some superfamily members are characterized as P-type ATPases because ATP-dependent transport is sensitive to the presence of vanadate. It has been widely postulated that CFTR hydrolyzes ATP to gate its chloride channel. However, direct evidence of CFTR hydrolytic activity in channel gating is lacking and existing circumstantial evidence is contradictory. Therefore, we evaluated CFTR chloride channel activity under conditions known to inhibit the activity of ATPases; i.e., in the absence of divalent cations and in the presence of a variety of ATPase inhibitors. Removal of the cytosolic cofactor, Mg2+, reduced both the opening and closing rates of CFTR suggesting that Mg2+ plays a modulatory role in channel gating. However, channels continued to both open and close showing that Mg2+ is not an absolute requirement for channel activity. The nonselective P-type ATPase inhibitor, vanadate, did not alter the gating of CFTR when used at concentrations which completely inhibit the activity of other ABC transporters (1 mM). Higher concentrations of vanadate (10 mM) blocked the closing of CFTR, but did not affect the opening of the channel. As expected, more selective P-type (Sch28080, ouabain), V-type (bafilomycin A1, SCN-) and F-type (oligomycin) ATPase inhibitors did not affect either the opening or closing of CFTR. Thus, CFTR does not share a pharmacological inhibition profile with other ATPases and channel gating occurs in the apparent absence of hydrolysis, although with altered kinetics. Vanadate inhibition of channel closure might suggest that a hydrolytic step is involved although the requirement for a high concentration raises the possibility of previously uncharacterized effects of this compound. Most conservatively, the requirement for high concentrations of vanadate demonstrates that the binding site for this transition state analogue is considerably different than that of other ABC transporters.
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PMID:Lack of conventional ATPase properties in CFTR chloride channel gating. 866 89

The rectal gland of the spiny dogfish shark, Squalus acanthias, secretes chloride by a furosemide sensitive process that has been termed "secondary active." Chloride enters the cell across the basolateral cell membrane via the sodium:potassium:2 chloride cotransporter. The energy for this electroneutral uptake step is provided by the electrochemical gradient for sodium directed into the cell. This is maintained by Na-K-ATPase present in the basolateral cell membrane. Present as well in the basolateral cell membrane is a potassium conductance that permits potassium to exit passively. Chloride leaves the cell across the luminal membrane via a chloride conductance closely similar to CFTR. The rectal gland is thus a model for the mechanism of secondary active chloride transport utilized by various epithelial organs throughout the vertebrate kingdom. This report reviews the humoral agents that regulate the secretion of chloride by the rectal gland and the intracellular mechanisms that mediate it. CNP, released from the heart in response to a volume stimulus, causes the release of VIP from nerves within the gland and together with VIP directly activates the rectal gland cell.
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PMID:The rectal gland of Squalus acanthias: a model for the transport of chloride. 874 53

The rectal gland of the dogfish shark (Squalus acanthias) is a sodium chloride secreting epithelial organ whose function was discovered in 1959 by Wendell Burger. The gland, composed of homogenous tubules of a single cell type, is an important model for secondary active chloride transport. Hormonal stimulation of chloride secretion in this system activates asymetrically arranged transport proteins (apical cAMP-activated CFTR-like Cl- channels, basolateral Na/K/2Cl cotransporters, Na/K-ATPase activity, and K+ channels). Five receptors, hormones, and membrane proteins of the shark rectal gland involved in chloride secretion have been cloned recently. Because the intact gland can be perfused via a single artery and vein, it has been possible to examine precisely the metabolic regulation of chloride transport by endogenous adenosine. Rectal gland cells have a high density of both stimulatory A2 type and inhibitory A1 type adenosine receptors. When stimulated by secretagogues, chloride secretion and venous adenosine concentrations increase in parallel, with chloride secretion increasing from approximately 150 to 2100 microEq/hr/g, and adenosine concentrations increasing from approximately 5 to approximately 890 nM. This work of ion transport is accompanied by a marked fall in intracellular ATP activity and a rise in both intracellular AMP and adenosine activity. Agents that prevent the interaction of endogenous adenosine with extracellular receptors significantly increase the chloride transport response to secretagogues. When chloride transport is inhibited by blocking the Na/K/2Cl cotransporter with bumetanide, both adenosine release and chloride secretion fall to basal values. We recently cloned a unique adenosine receptor subtype that is distinct from previously cloned mammalian adenosine receptors. Because of its highly specialized function, single cell type, and simple vascular system, the shark rectal gland is an ideal model system for examining the metabolic regulation of chloride secretion by adenosine receptors.
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PMID:Cellular and molecular biology of chloride secretion in the shark rectal gland: regulation by adenosine receptors. 874 54

Regulation of intracellular pH (pHi) was studied in cultured bovine tracheal epithelial cells using microspectrofluorimetry of the fluorescent indicator 2',7'-biscarboxyethyl- 5(6)-carboxyfluorescein (BCECF). The cells, which were grown on coverslips and superfused in a chamber on the stage of a microscope, were acidified by NH4Cl-prepulses, and pHi recovery was measured (in DeltapH/min) at approximately pHi 6.7. In HCO3-free solutions the recovery rate was 0.14 pH/min, and addition of amiloride or Na-free solution reduced this rate to 0.02-0.03 pH/min. In HCO3/CO2-buffered Ringer's, the rate of recovery was 0.32 pH/min, and amiloride or Na-free reduced the rate to 0.08-0.10 pH/min. This residual Na-independent and HCO3-dependent pHi recovery was studied by using inhibitors of HCO3 and H transporters. Bafilomycin (inhibits H-ATPases) at 100 nM did not significantly affect pHi recovery, while 100 microM SCH28080 (inhibits H,K-ATPase) had a variable inhibitory effect (25-75%), indicating that a gastric-like H, K-ATPase, but not electrogenic H pump, may contribute in a minor way to the recovery from acidification. Cl-free solution and 500 microM H2DIDS (dihydro-4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, blocks anion exchange and the outwardly rectifying Cl channel, ORCC), both blocked apparent anion exchange activity, but had no effect on the recovery; 100 microM DNDS (4-4''-dinitro-2-2'-stilbenedisulfonate blocks the ORCC but not the cystic fibrosis transmembrane conductance regulator, CFTR) had no effect on pHi recovery; DPC (diphenylamine carboxylate, blocks the CFTR and the ORCC) caused a complete and reversible inhibition of the recovery. When [K] was increased ten fold to depolarize the cell's membrane potential, the magnitude of the pHi recovery (though not the rate) was enhanced. Thus, the HCO3-dependent, Na- and Cl-independent, DPC-blockable pHi recovery may be largely due to an influx of HCO3 via CFTR Cl channels. Under physiological conditions, when the electrochemical gradient for HCO3 is likely to be outwardly rather than inwardly directed, the CFTR (or another HCO3-permeable channel) may mediate HCO3 secretion and contribute to regulation of pH of the periciliary fluid.
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PMID:HCO3-dependent pHi regulation in tracheal epithelial cells. 876 16

CFTR-NBF-2 expressed and purified in fusion with the maltose-binding protein was shown to catalyse the reaction ATP-->ADP+Pi by three different assays, monitoring ATP turnover, formation of ADP and release of Pi (Km 86 microM, rate constant 0.37 min(-1)). The reaction product ADP inhibits this ATPase activity. In a similar manner the hydrolysis of GTP to GDP and Pi was demonstrated (Km 40 microM, rate constant 0.29 min(-1)). In the presence of AMP the ATPase reaction was superseded by the formation of two ADP from ATP and AMP. As typical for adenylate kinases a distinct AMP-binding site could be verified for CFTR-NBF-2 by the inability of TNP-ATP and AMP to compete for binding. All three enzymatic activities were inhibited by the symmetric double-substrate-mimicking inhibitor Ap5A. As NBF-2 plays a central role in CFTR channel opening and closing the results reported here are fundamental in understanding mechanisms of CFTR channel activity regulation.
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PMID:A recombinant polypeptide model of the second nucleotide-binding fold of the cystic fibrosis transmembrane conductance regulator functions as an active ATPase, GTPase and adenylate kinase. 923 25


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