Molecular characterization of the epithelial Na-K-Cl cotransporter isoforms.

Recent advances in the molecular characterization of specific isoforms of the Na-K-Cl cotransporter have allowed rapid progress in the study of the structure, function, and regulation of these members of a family of Cl-dependent cation cotransporters. Two distinct isoforms have been identified, one from Cl(-)-secretory epithelia and another found specifically in the diluting segment of the vertebrate kidney, a Cl(-)-absorptive epithelium. The discovery of three alternatively spliced variants of the absorptive isoform, which differ only by 31 amino acids and which appear to be differentially distributed within the mammalian thick ascending limb of the loop of Henle, highlight this spliced region as an important functional component of the protein.

Molecular cloning and immunological characterization of the gamma polypeptide, a small protein associated with the Na,K-ATPase.

The gamma subunit of the Na,K-ATPase is a small membrane protein that copurifies with the alpha and beta subunits of the enzyme. Strong evidence that the gamma subunit is a component of the Na,K-ATPase comes from studies indicating that the subunit is involved in forming the site for cardiac glycoside binding. We have isolated and characterized the cDNAs coding the gamma subunit from several species. The gamma subunit is a highly conserved protein consisting of 58 amino acids with a molecular weight of 6500. Hydropathy analysis reveals the presence of a single hydrophobic domain that is sufficient to cross the membrane. There are no sites for N-linked glycosylation. Northern blot analysis revealed that the gamma subunit mRNA is expressed in a tissue-specific fashion and is present in all tissues characterized. gamma-specific antibodies have been used to verify that the sequenced protein is the same protein labeled by [3H]nitroazidobenzoyl-ouabain (NAB-ouabain), and that this protein, the gamma subunit of the Na,K-ATPase, has a distribution pattern along nephron segments that is identical with the alpha subunit. In addition, coimmunoprecipitation of the alpha, beta and gamma subunits demonstrate specific association of the subunits. These results are consistent with the notion that the gamma subunit is specifically associated with and may be an important component of the Na,K-ATPase.

Photoaffinity labelling of a 150 kDa (Na + K + Cl)-cotransport protein from duck red cells with an analog of bumetanide.

We have used a radiolabelled, benzophenone analog of bumetanide, 4-[3H]benzoyl-5-sulfamoyl-3-(3-thenyloxy)benzoic acid ([3H]BSTBA) to photolabel plasma membranes from duck red blood cells. BSTBA, like bumetanide, is a loop diuretic and a potent inhibitor of (Na + K + Cl) cotransport, and [3H]BSTBA binds to intact duck red cells with a high affinity similar to that of [3H]bumetanide (K 1/2 congruent to 0.1 microM). We incubated duck red cells with [3H]BSTBA, then lysed the cells and exposed the ghosts to ultraviolet light. The ghosting and photolysis was done at 0 degree C to prevent dissociation of the [3H]BSTBA. The ghosts were then sonicated to remove the nuclei and run on SDS-polyacrylamide gels. Analysis of H2O2-digested gel slices revealed [3H]BSTBA to be incorporated into a protein of approx. 150 kDa. This is the same molecular weight we obtain for a protein from dog kidney membranes which is photolabelled by [3H]BSTBA in a manner highly consistent with labelling of the (Na + K + Cl) cotransporter (Haas and Forbush (1987) Am. J. Physiol. 253, C243-C252). Several lines of evidence strongly suggest that the 150 kDa protein from duck red cell membranes is an integral component of the (Na + K + Cl)-cotransport system in these cells: (1) Photolabelling of this protein by [3H]BSTBA is blocked when 10 microM unlabelled bumetanide is included in the initial incubation medium with [3H]BSTBA; (2) Photoincorporation of [3H]BSTBA into the 150 kDa protein is markedly increased when the initial incubation medium is hypertonic or contains norepinephrine, conditions which similarly stimulate both (Na + K + Cl) cotransport and saturable [3H]bumetanide binding in duck red cells; (3) The photolabelling of this protein shows a saturable dependence on [3H]BSTBA concentration, with a K1/2 (0.06 microM) similar to that for the reversible, saturable binding of [3H]BSTBA and [3H]bumetanide to duck red cells; and (4) [3H]BSTBA photoincorporation into the 150 kDa protein, like saturable [3H]bumetanide binding to intact cells, requires the simultaneous presence of Na+, K+, and Cl- in the medium containing the radiolabelled diuretic.

Direct photoaffinity labeling of the primary region of the ouabain binding site of (Na+ + K+)-ATPase with [3H]ouabain, [3H]digitoxin and [3H]digitoxigenin.

The tritiated cardiotonic steroids, ouabain, digitoxin, and digitoxigenin are shown to photolabel the large polypeptide but not the glycoprotein or proteolipid component of the (Na+ + K+)-ATPase when they are bound to the inhibitory site and exposed to light of 220 or 254 nm. The extent of photolabeling is low, less than 1%, and is limited by photocross-linking of the enzyme. The mechanism of photoincorporation does not appear to be either photolysis of the lactone ring in ouabain or photolysis of tryptophan or tyrosine residues in the polypeptide.

Purification and characterization of an (Na+ + K+)-ATPase proteolipid labeled with a photoaffinity derivative of ouabain.

Highly purified lamb kidney (Na+ + K+)-ATPase was photoaffinity labeled with the tritiated 2-nitro-5-azidobenzoyl derivative of ouabain (NAB-ouabain). The labeled (Na+ + K+)-ATPase was mixed with unlabeled carrier enzyme. Two proteolipid (gamma 1 and gamma 2) fractions were then isolated by chromatography on columns of Sepharose CL-6B and Sephadex LH-60. The two fractions were interchangeable when rechromatographed on the LH-60 column, suggesting that gamma 1 is an aggregated form of gamma 2. The total yield was 0.8-1.5 mol of gamma component per mol of catalytic subunit recovered. This indicates that the gamma component is present in stoichiometric amounts in the Na+ + K+)-ATPase. The proteolipids that were labeled with NAB-ouabain copurified with the unlabeled proteolipids.

Mutagenic mapping of the Na-K-Cl cotransporter for domains involved in ion transport and bumetanide binding.

The human and shark Na-K-Cl cotransporters (NKCCs) are 74% identical in amino acid sequence yet they display marked differences in apparent affinities for the ions and bumetanide. In this study, we have used chimeras and point mutations to determine which transmembrane domains (tm's) are responsible for the differences in ion transport and in inhibitor binding kinetics. When expressed in HEK-293 cells, all the mutants carry out bumetanide-sensitive 86Rb influx. The kinetic behavior of these constructs demonstrates that the first seven tm's contain all of the residues conferring affinity differences. In conjunction with our previous finding that tm 2 plays an important role in cation transport, the present observations implicate the fourth and seventh tm helices in anion transport. Thus, it appears that tm's 2, 4, and 7 contain the essential affinity-modifying residues accounting for the human-shark differences with regard to cation and anion transport. Point mutations have narrowed the list of candidates to 13 residues within the three tm's. The affinity for bumetanide was found to be affected by residues in the same tm 2-7 region, and also by residues in tm's 11 and 12. Unlike for the ions, changes in bumetanide affinity were nonlinear and difficult to interpret: the Ki(bumetanide) of a number of the constructs was outside the range of sNKCC1 and hNKCC1 Kis.

Rapid 86Rb release from an occluded state of the Na,K-pump reflects the rate of dephosphorylation or dearsenylation.

The rapid release of 86Rb from an occluded state of the Na,K-ATPase was studied in a rapid filtration apparatus. In the presence of Mg2+, 86Rb release was found to be stimulated by arsenate and thiophosphate just as it is stimulated by Pi. The affinity of Na,K-ATPase for arsenate is about 4-fold higher than that for Pi and the affinity for thiophosphate is about 5-fold lower than that for Pi. With all three divalent anions, the rates of maximally stimulated 86Rb release were constant between pH 6.5 and 7.5, and decreased between pH 7.5 and 8.5. The affinity for phosphate and thiophosphate increased in the latter pH range, while the affinity for arsenate decreased. The results are not consistent with titration of the divalent anion as the sole determinant of effectiveness in stimulating 86Rb release; thus they suggest that titration of groups on the protein is important. A delay in the rise to the maximum rate of 86Rb release upon stimulation with arsenate is shown to be due to the time required for arsenylation, and from an analysis of the rise and fall of the rate of 86Rb release the rate constants for arsenylation and dearsenylation at pH 7.2 can be estimated; the decay in the rate of 86Rb release when arsenate or phosphate is removed from the solution provides a second method for determination of the dearsenylation rate. The dearsenylation rate constant increases 5-fold from pH 6.1 to 7.5. From the time course of 86Rb release in the presence of Pi we estimate that the rate of dephosphorylation is 50-100 s-1 at pH 6.6 and 20 degrees C; at pH 7-7.5 the rate is too fast to determine. Dimethyl sulfoxide (25%) increases the affinity for arsenate (or phosphate), due to reciprocal changes in arsenylation and dearsenylation rates, and it increases the rate of 86Rb release 2-3 fold. Finally, the level of phosphointermediate formation from 32Pi was determined in the absence and presence of K+: when methanol is used as a denaturant, K+ has only a small effect on the observed level of E-32P, but when trichloroacetic acid is used, K+ is found to reduce the observed level to less than 50% of the control value.

Rapid release of 45Ca from an occluded state of the Na,K-pump.

45Ca is bound to the occluded state of the Na,K-pump, apparently at K+ sites. Only one 45Ca ion is bound in place of two K+ ions, with an affinity approximately 0.08 mM; K+ competes with an apparent affinity approximately 0.04 mM. 45Ca is released rapidly from Na,K-ATPase in the presence of ATP or ADP, presumably to the intracellular medium. The rate constant of 45Ca release with ATP is greater than 100 s-1 at 20 degrees C, more than twice as fast as the rate of release of 42K from the occluded state. Phosphorylation of Na,K-ATPase with MgPi, which would lead to release of occluded K+ or Rb+ to the extracellular face of the membrane, stabilizes occluded 45Ca. 45Ca release is slower immediately after exposure to MgPi than after a rinse in the absence of Pi indicating that in the former circumstance the rate of 45Ca release is limited by dephosphorylation; 45Ca release is even slower after exposure to Mg2+ arsenate, consistent with dearsenylation being slower than dephosphorylation. When limited by dephosphorylation, the rate of 45Ca release is dependent on the species of monovalent cation present, increasing in the order N-methylglucamine less than Cs+ less than Li+ less than Na+ less than Rb+ less than K+. When the 45Ca occluded state is exposed to K + Mg + Pi and then to Na+ + Mg2+ + ATP, the exposure to K+ is "remembered," indicating simultaneous occlusion of 45Ca and K+. The apparent affinity for K+ in formation of this state is 10-50 mM, and the rate of release of K+ is approximately 2 s-1. Ca2+ has effects on the release of 86Rb from the occluded state: With ATP, Ca2+ acts like Mg2+ by stimulating 86Rb release at low concentrations and inhibiting at high concentrations; with MgPi, Ca2+ inhibits 86Rb release, presumably by preventing phosphorylation. Thus, Ca2+ has two actions on the Na,K-pump as studied here: one as a Mg2+ congener, and another as a K+ congener at transport sites. In the latter role Ca2+ is unusual in that it appears to be able to bind to the transport sites from the intracellular face of the pump and to become occluded, but unable to be released from extracellular sites.

The interaction of amines with the occluded state of the Na,K-pump.

We have studied the effect of various amines on the rate of release of 86Rb from the occluded state of dog kidney Na,K-ATPase formed by pre-incubation of the enzyme with 86Rb. In the presence of MgPi, various amines act like K+ or Rb+ in blocking the release of 86Rb from one of two sites for occlusion (the "s" site). Of 38 amines tested, tetrapropylamine and various benzyl amines exhibit the highest affinity; the K1/2 for these compounds is 2-5 mM. In the presence of ATP, when 86Rb is presumably released towards the intracellular face of the pump in the normal mode of operation, 86Rb release is blocked by the presence of amine, but only if the amine is also included in a preincubation with MgPi. The data are consistent with a model in which the interaction of amine with one of the transport sites (the "f" site) prevents the E2----E1 transformation that is stimulated by ATP. When 86Rb deocclusion from the f site has occurred in the presence of amine, the lone 86Rb at the s site can be released in the presence of ATP if the amine is removed from the medium. This suggests that a single 86Rb ion at the s site can be released to the intracellular face of the membrane, and therefore that transport can occur with only one K+ site occupied. The amine that blocks release of one 86Rb ion does not itself become occluded: (a) The interaction of amine and ATP is only seen when both ligands are present in the medium; (b) the effects of amines are not "remembered" after a brief exposure to a rinse medium; (c) with the vanadate-inhibited enzyme, benzyltriethylamine and tetrapropylamine are only weakly effective in blocking 86Rb release from the s site; and (d) organic cations exhibit very low affinity in competition with 86Rb for occlusion at equilibrium. Thus the results are consistent with the idea that monofunctional amines block by binding to the f site but that, unlike K+ and Rb+, they do not become occluded. In contrast, at equilibrium ethylenediamine prevents 86Rb occlusion in a competitive manner, suggesting the possibility of occlusion of the bifunctional amine.

Rapid release of 42K and 86Rb from an occluded state of the Na,K-pump in the presence of ATP or ADP.

We have measured the time course of release of 42K and 86Rb from an occluded state of the Na,K-pump using a rapid filtration apparatus. We have found that at 20 degrees C and in the presence of ATP, 42K is released with a rate constant of approximately 45 s-1 and 86Rb with a rate constant of approximately 20 s-1; both ATP and ADP are effective at a low affinity site (Kd approximately 0.3 and 1 mM, respectively) with the rate of deocclusion being only half as great in ADP as in ATP. Mg2+ stimulates 2-fold at low concentrations probably by forming MgATP, and free Mg2+ is strongly inhibitory at high concentrations (Kd approximately 10 mM). Mg2+ also decreases the affinity for ATP, and the data are consistent with mixed type inhibition; from the analysis the dissociation constant is approximately 1 mM for the inhibitory Mg2+ and the Rb+-occluded form without ATP. The rate of 42K or 86Rb release increases monotonically with pH while ATPase activity decreases above pH 8, so that deocclusion is not rate-limiting in the overall cycle at high pH. This is reflected by a convergence of the rate of Na,K-ATPase and Na,Rb-ATPase activities at high pH and by a decrease in the observed steady-state level of the occluded 86Rb intermediate at high pH. K+, Rb+, Na+, and Cs+, but not Li+, increase the rate of 42K and 86Rb release at constant ionic strength, presumably at sites other than the transport sites. The spontaneous rate of deocclusion is only approximately 0.1 s-1 at low ionic strength in the absence of nucleotides, and it is increased markedly by all cations tested except Li+. Overall the data are consistent with deocclusion as a rate-limiting step in the Na,K-pump cycle.

Rapid release of 42K or 86Rb from two distinct transport sites on the Na,K-pump in the presence of Pi or vanadate.

The rate of 86Rb or 42K release from an occluded form of the phosphorylated Na+ pump has been studied using a rapid filtration apparatus described previously. The rate constant of release is 5-15 s-1, and 42K and 86Rb dissociate at approximately the same rate. Mg2+ is required for deocclusion in the presence of Pi at a site which has the same affinity as the site involved in stabilization of E2(K) with ATP; we propose that Na,K-ATPase has only one site for Mg2+ (apart from Mg2+ complexed with ATP), that the affinity of this site for Mg2+ is increased by Pi binding and decreased by ATP binding, and that Mg2+ is bound and released in the normal transport cycle. In the presence of K+, Cs+, Rb+, or Tl+, the release of two distinct 86Rb ions can be observed, the slow release from one site ("s" site) being blocked by occupancy of the site vacated by the other ("f", fast site). By a sequence of incubations, labeled 86Rb can be placed at either site, and the rate of dissociation monitored individually; in the absence of K+, dissociation from the s site proceeds after a lag in which the f site is vacated. The results are consistent with a "flickering-gate" model of deocclusion to the extracellular pump face, in which the site is exposed to the medium only long enough for a single ion to be released. When deocclusion to the intracellular face is promoted with ATP, ions are released from both sites at the same rate, presumably because the E2----E1 conformational change is rate-limiting. Unlabeled ions co-occluded with 86Rb increase the ATP-stimulated rate of release in the order Rb+ less than Tl+ less than Cs+ less than K+; since the same rank order is observed when dissociation from the s site is monitored in the presence of these ions and MgPi we propose that the latter process proceeds toward the intracellular pump face. 86Rb release from the vanadate-inhibited enzyme has the characteristics of Pi-stimulated release but is approximately 25-fold slower. ATP binds to both the phosphorylated and vanadate-inhibited forms of Na,K-ATPase and increases the rate of deocclusion, apparently to both the intracellular and extracellular faces of the pump.

Assay of Na,K-ATPase in plasma membrane preparations: increasing the permeability of membrane vesicles using sodium dodecyl sulfate buffered with bovine serum albumin.

Determination of maximal Na,K-ATPase activity in isolated plasma membranes is generally hampered by the vesicular nature of the preparation, limiting access of ATP and ions to one face or the other of the transmembrane protein. Detergents are often used to make the vesicles permeable to the substrates; however, the detergent/protein ratio is extremely critical for optimal activation. The use of bovine serum albumin as a detergent buffer is described. With this method the amount of membrane protein in the assay can be varied over a wide range, with full detergent activation. The method has been used for assay of Na,K-ATPase activity of membranes from dog kidney, rabbit brain, and electric organ of eel.

Sodium and potassium fluxes across the dialyzed giant axon of Myxicola.

Resting and stimulated fluxes of sodium and potassium across the giant axon of the marine annelid, Myxicola infundibulum, have been characterized using the technique of internal dialysis. In most respects the ion movements were found to be similar to those in squid axons. Sodium efflux and potassium influx were found to be active, cardiac glycoside-sensitive fluxes, with a variable coupling ratio. However, when [ATP]i was lowered to less than 20 microM by treatment with cyanide and continuous dialysis, or to less than 2 microM by dialysis with glucose following injection of hexokinase, Na efflux and K influx were unaltered. The maintained fluxes were not accounted for by an increased passive permeability of the axolemma, although 30-60% of the Na efflux appeared to be due to Na-Na exchange. An altered form of Na pump operation at low [ATP]i is a more likely explanation than an alternate energy source, or an ATP source proximate to the axolemma. The transient response of 22Na efflux to a change in [22Na]i was found to be much slower than in squid, tau = 360 sec. The efflux delay could only be accounted for by an extra-axonal diffusion barrier, which is probably the basement membrane surrounding the ventral nerve cord.

An apparatus for rapid kinetic analysis of isotopic efflux from membrane vesicles and of ligand dissociation from membrane proteins.

A new method for the measurement of rapid isotopic release from a membrane compartment is described. Membrane vesicles loaded with isotope, or broken membranes with bound radioactive ligand, are filtered onto the surface of a cellulose ester filter; the rate of the loss of isotope from the membrane compartment is followed continuously by collecting fluid which is passed through the filters under high pressure. A change in release rate is initiated by changing the solution or by delivering a flash of light to a photosensitive sample. The approach has been used to study rapid 22Na efflux from membrane vesicles rich in the ouabain-sensitive Na pump, and to examine dissociation of 32P and 86Rb from membrane-bound Na,K-ATPase. Since the rate of efflux is measured, and not the total counts remaining on the filter, the technique has high sensitivity. A complete time course is obtained using only a few micrograms of membrane protein. The apparatus described is simple, inexpensive, and easily constructed; with the present device, time resolution is approximately 10 ms.

Na+ movement in a single turnover of the Na pump.

Ouabain-sensitive 22Na efflux from right-side-out membrane vesicles prepared from dog kidney has been examined with a time resolution of 30 msec. The vesicles are preloaded with 22Na and caged ATP [P3-1-(2-nitro)phenylethyl adenosine triphosphate], so that transport by the Na pump can be initiated by light. After a brief illumination, which releases less ATP than the number of catalytic sites, a burst of 22Na extrusion is observed corresponding to a single turnover of the Na pump. By the use of a rapid filtration apparatus, with which a continuous record of the rate of efflux is obtained, it has been possible to resolve the efflux burst in the time range of 20-1500 msec. The rate of efflux rises rapidly, but not instantaneously, to a peak and then decays, with a time constant of approximately equal to 6 sec-1 at 15 degrees C. The time course of Na efflux is unaffected by extracellular K+, as predicted by models of the Na pump in which Na is released early in the cycle. Unphotolyzed caged ATP is found to bind to the catalytic site of Na,K-ATPase, in competition with ATP that is produced in the flash, and the possibility has not been excluded that dissociation of unphotolyzed caged ATP and binding of ATP are involved in the Na efflux time course. It seems most likely that binding of ATP and translocation of 22Na are involved in the increase in the 22Na efflux rate in the single turnover and that the release of transported 22Na from extracellular pump sites limits the slow decay.

Characterization of right-side-out membrane vesicles rich in (Na,K)-ATPase and isolated from dog kidney outer medulla.

When purified on a sucrose gradient, basolateral membranes from dog kidney outer medulla are found to be very rich in (Na,K)-ATPase; about 50% of the membrane protein is comprised of this enzyme. (Na,K)-ATPase activity is activated 3- to 5-fold by detergent treatment, and this has been previously attributed to the impermeable vesicular nature of the membranes. Porcine trypsin inactivates only that fraction of (Na,K)-ATPase activity seen without detergent, consistent with a right-side-out orientation of membrane vesicles; the trypsin sensitivity and detergent activation of [3H]ouabain binding in the presence of Na+ + Mg2+ + ATP or Mg2+ + Pi are also consistent with this hypothesis. Using nearly isosmotic Hypaque density gradient centrifugation a population of impermeable right-side-out membrane vesicles (H1) is separated from a leaky population (H2). (Na,K)-ATPase activity in the H1 population is 20-fold activated by detergent and insensitive to porcine trypsin. The vesicle volume is 2.4 microliters/mg, and monovalent cations passively equilibrate with the intravesicular volume on a time scale of 5-30 min. Very rapid ouabain sensitive 22Na efflux from the vesicles is observed when ATP is photolytically released from intravesicular caged ATP.