The effect of the gamma modulator on Na/K pump activity of intact mammalian cells.

This study concerns the modulatory effects of the gamma modulator of the Na/K pump, in particular whether the effects seen in previous experiments with isolated membranes are relevant to Na/K pump behavior in intact mammalian cells. For this purpose, HeLa cells previously transfected with the rat Na/K catalytic subunit were used. The results show that both variants of the regulator, gammaa and gammab, decrease the apparent affinity of the pump for Na(+) and cause a modest increase in apparent ATP affinity as seen in measurements of ouabain-sensitive (86)Rb(K(+)) influx into cells in which ATP was varied using antimycin A and glucose. Equivalent results had been obtained previously in our analyses of Na,K-ATPase activity of membrane fragments, i.e., an increase in K(0.5(Na)) at high K(+) concentration and a decrease in K'(ATP). Comparison of clones of gamma-transfected and mock-transfected cells (with similar V(max) values) indicated that gamma causes a modest approximately 30% increase in the steady-state concentration of intracellular Na(+). Furthermore, for both gammaa and gammab, values of intracellular Na(+) were similar to those predicted from the kinetic constants, K(0.5(Na)) and V(max). Finally, there was a gamma-mediated increase in apparent affinity for extracellular K(+), which had not been detected in assays of permeabilized membranes.

The KCl cotransporter isoform KCC3 can play an important role in cell growth regulation.

The KCl cotransporter (KCC) plays a significant role in the ionic and osmotic homeostasis of many cell types. Four KCC isoforms have been cloned. KCC1 and KCC4 activity is osmolality-sensitive and involved in volume regulation. KCC2, a neuronal-specific isoform, can lower intracellular Cl(-) and is critical for inhibitory GABA responses in the mature central nervous system. KCC3, initially cloned from vascular endothelial cells, is widely but not universally distributed and has an unknown physiological significance. Here we show a tight link between the expression and activity of KCC3 and cell growth by a NIH/3T3 fibroblast expression system. KCC3 activity is sensitive to [(dihydroindenyl)oxy] alkanoic acid (DIOA) and N-ethylmaleimide and is regulated by tyrosine phosphorylation. Osmotic swelling does not activate KCC3, and the process of regulatory volume decrease is refractory to DIOA, indicating that KCC3 is not involved in volume regulation. KCC3 expression enhances cell proliferation, and this growth advantage can be abolished by the inhibition of KCC3 by DIOA. Fluorescence-activated cell sorting measurements and Western blot analysis show DIOA caused a significant reduction of the cell fraction in proliferative phase and a change in phosphorylation of retinoblastoma protein (Rb) and cdc2, suggesting that KCC3 activity is important for cell cycle progression. Insulin-like growth factor-1 up-regulates KCC3 expression and stimulates cell growth. Tumor necrotic factor-alpha down-regulates KCC3 expression and causes growth arrest. These data indicate that KCC3 is an important KCC isoform that may be involved in cell proliferation.

Candidate inhibitor of the volume-sensitive kinase regulating K-Cl cotransport: the myosin light chain kinase inhibitor ML-7.

K-Cl cotransport, KCC, is activated by swelling in many cells types, and promotes volume regulation by a KCl efflux osmotically coupled to water efflux. KCC is probably activated by swelling-inhibition of a kinase, permitting dephosphorylation, and activation of the cotransporter by a phosphatase. The myosin light chain kinase (MLCK) inhibitor ML-7 inhibits transporters activated by shrinkage. In red blood cells from three mammalian species, ML-7 stimulated KCC in a volume-dependent manner. Relative stimulation was greatest in more shrunken cells. Stimulation was reduced by moderate cell swelling and abolished by further swelling. The half-maximal stimulation is at approximately 20 microm ML-7, 50-fold greater than the IC(50) for inhibition of MLCK in vitro. Stimulation of KCC by ML-7 did not require cell Ca, while MLCK does. Therefore the target of ML-7 in stimulating KCC in red cells is probably not MLCK. The evidence favors stimulation of KCC by ML-7 by inhibiting the volume-sensitive kinase. Qualitatively similar effects of ML-7 on KCC in red cells from three mammalian species suggest a general mechanism.

Molecular cloning and functional characterization of KCC3, a new K-Cl cotransporter.

We isolated and characterized a novel K-Cl cotransporter, KCC3, from human placenta. The deduced protein contains 1,150 amino acids. KCC3 shares 75-76% identity at the amino acid level with human, pig, rat, and rabbit KCC1 and 67% identity with rat KCC2. KCC3 is 40 and 33% identical to two Caenorhabditis elegans K-Cl cotransporters and approximately 20% identical to other members of the cation-chloride cotransporter family (CCC), two Na-K-Cl cotransporters (NKCC1, NKCC2), and the Na-Cl cotransporter (NCC). Hydropathy analysis indicates a typical KCC topology with 12 transmembrane domains, a large extracellular loop between transmembrane domains 5 and 6 (unique to KCCs), and large NH(2) and COOH termini. KCC3 is predominantly expressed in kidney, heart, and brain, and is also expressed in skeletal muscle, placenta, lung, liver, and pancreas. KCC3 was localized to chromosome 15. KCC3 transiently expressed in human embryonic kidney (HEK)-293 cells fulfilled three criteria for increased expression of K-Cl cotransport: stimulation of cotransport by swelling, treatment with N-ethylmaleimide, or treatment with staurosporine.

Cloning, characterization, and gene organization of K-Cl cotransporter from pig and human kidney and C. elegans.

We isolated and characterized the cDNAs for the human, pig, and Caenorhabditis elegans K-Cl cotransporters. The pig and human homologs are 94% identical and contain 1,085 and 1,086 amino acids, respectively. The deduced protein of the C. elegans K-Cl cotransporter clone (CE-KCC1) contains 1,003 amino acids. The mammalian K-Cl cotransporters share approximately 45% similarity with CE-KCC1. Hydropathy analyses of the three clones indicate typical KCC topology patterns with 12 transmembrane segments, large extracellular loops between transmembrane domains 5 and 6 (unique to KCC), and large COOH-terminal domains. Human KCC1 is widely expressed among various tissues. This KCC1 gene spans 23 kb and is organized in 24 exons, whereas the CE-KCC1 gene spans 3.5 kb and contains 10 exons. Transiently and stably transfected human embryonic kidney cells (HEK-293) expressing the human, pig, and C. elegans K-Cl cotransporter fulfilled two (pig) or five (human and C. elegans) criteria for increased expression of the K-Cl cotransporter. The criteria employed were basal K-Cl cotransport; stimulation of cotransport by swelling, N-ethylmaleimide, staurosporine, and reduced cell Mg concentration; and secondary stimulation of Na-K-Cl cotransport.

Stimulation of membrane serine-threonine phosphatase in erythrocytes by hydrogen peroxide and staurosporine.

Indirect evidence has suggested that K-Cl cotransport in human and sheep erythrocytes is activated physiologically by a serine-threonine phosphatase. It is activated experimentally by H2O2 and by staurosporine, a kinase inhibitor. Activation by H2O2 and staurosporine is inhibited by serine-threonine phosphatase inhibitors, suggesting that the activators stimulate the phosphatase. The present study shows that sheep and human erythrocytes contain membrane-associated as well as cytosolic serine-threonine phosphatases, assayed from the dephosphorylation of 32P-labeled glycogen phosphorylase. In cells from both species, the relatively low sensitivity of the membrane enzyme to okadaic acid suggests it is type 1 protein phosphatase. The cytosolic phosphatase was much more sensitive to okadaic acid. Membrane-associated phosphatase was stimulated by both H2O2 and staurosporine. The results support earlier conclusions that the membrane-associated type 1 phosphatase identified here is regulated by phosphorylation and oxidation. The results are consistent with the phosphatase, or a portion of it, being responsible for activating K-Cl cotransport.

L antigens of sheep red blood cell membranes and modulation of ion transport.

Sheep are polymorphic with respect to the intracellular Na+ and K+ concentrations of their erythrocytes. Erythrocytes of sheep of the high-K+ (HK) phenotype have high K+ and low Na+ concentrations; erythrocytes from sheep of the allelic low-K+ (LK) phenotype have abnormally low K+ and high Na+ concentrations. The difference is due to differences in rates of cation transport: higher Na+-K+ pump flux in HK cells and higher K+-Cl- cotransport in LK cells. The HK/LK polymorphism is associated with a polymorphism of red blood cell antigens: the L antigen is only on LK cells, and HK cells have only the M antigen. There are two classes of L antigen that assort together: Lp, which is associated with Na+-K+ pumps, and Ll, which is associated with K+-Cl- cotransporters. There are functional consequences of these associations: anti-Lp antibody stimulates the pump and anti-Ll antibody inhibits cotransport. The use of these antibodies has permitted delineation of the roles of the antigens in modulating the function of the transporters. In this review, we summarize the evidence that these antigens are entities distinct from the pump. The Lp antigen reacts reversibly with the Na+-K+ pump; the antigen inhibits the pump, mainly by promoting nonspecific inhibition by intracellular K+. The antigen also modulates pump differentiation in immature cells. In contrast, the Ll antigen stimulates K+-Cl- cotransport. The evidence suggests that the two polymorphisms are controlled by a single genetic locus and that all of the distinct properties of ion transporters in LK cells are attributable to interactions with L antigens.

Mechanism of swelling activation of K-Cl cotransport in inside-out vesicles of LK sheep erythrocyte membranes.

Stimulation by swelling of K-Cl cotransport was studied in inside-out vesicles (IOVs) made from membranes of LK sheep erythrocytes. The purpose was to understand this stimulation in terms of the three-state process proposed for regulation of the cotransporter (P.B. Dunham, J. Klimczak, and P.J. Logue. J. Gen. Physiol. 101: 733-765, 1993). The first step in this process, A --> B, is rate limiting and controlled by transphosphorylation reactions. The second step, B --> C, is fast; its control is unknown. Predictions were that maximum velocity (Jmax) of cotransport increases with A --> B and concentration at one-half Jmax (K1/2) of K+ as a substrate decreases with B --> C. We tested the hypothesis that most transporters in IOVs are in the B state and that swelling activates cotransport in vesicles by the B --> C conversion. In accordance with this hypothesis, swelling should activate K+ influx with no discernable delay. It did. K1/2 for K+ should decrease with swelling and Jmax should not change. K1/2 decreased 10-fold, and Jmax did not change. Inhibitors of transphosphorylation, reactions of A --> B, should not affect K+ flux into IOVs, and they did not. The results support the hypothesis: swelling activation of K+ flux into IOVs corresponds to B --> C. A mechanical change in the membrane causes a specific change in the cotransporter: an increase in apparent affinity for K+.

Reconstitution of calyculin-inhibited K-Cl cotransport in dog erythrocyte ghosts by exogenous PP-1.

Osmotic swelling of dog and other mammalian erythrocytes activates Cl-dependent K transport, K-Cl cotransport. This activation can be abolished by nanomolar concentrations of calyculin, a potent inhibitor of serine-threonine protein phosphatases. Therefore, K-Cl cotransport is probably activated by dephosphorylation by a type 1 and/or type 2A protein phosphatase (PP-1 and PP-2A, respectively). This was tested directly by incorporating exogenous protein phosphatases into resealed ghosts made from dog erythrocytes previously exposed to calyculin. K-Cl cotransport was nearly completely inhibited in the ghosts. Incorporation of PP-1 reconstituted K-Cl cotransport. Maximal reconstitution was up to 90% of the control flux in the ghosts and 0.1 U PP-1/ml lysate gave half-maximal reconstitution of cotransport. In contrast, PP-2A had no effect. This result with PP-1 provides direct evidence that K-Cl cotransport is activated by PP-1 in dog erythrocytes. Half-maximal activation of K-Cl cotransport required approximately 180 molecules of PP-1 per ghost.

H2O2 activates red blood cell K-Cl cotransport via stimulation of a phosphatase.

K-Cl cotransport is involved in volume regulation in a number of cell types. Cell swelling stimulates K-Cl cotransport, probably by inhibition of a volume-sensitive kinase. K-Cl cotransport can also be activated by oxidants and thiol reagents. We investigated the effect of H2O2 on K-Cl cotransport of LK sheep red blood cells in an attempt to identify the target of oxidants. H2O2 stimulated K-Cl cotransport. The stimulation was virtually abolished by subsequent incubation with calyculin, a protein phosphatase inhibitor. This suggests that H2O2 stimulates a calyculin-sensitive phosphatase and activates K-Cl cotransport by causing a decrease in phosphorylation of the transporter or a regulatory protein. The thiol reagent N-ethylmaleimide, which stimulates K-Cl cotransport, did not stimulate cotransport further in cells with cotransport activated by staurosporine but did stimulate cotransport further in cells with cotransport activated by H2O2. These results suggest that there are at least two distinct phosphorylation sites on the transporter or a regulator. The results also suggest that the phosphatase is associated with the membrane.

Effects of ionic strength on the regulation of Na/H exchange and K-Cl cotransport in dog red blood cells.

Dog red cell membranes contain two distinct volume-sensitive transporters: swelling-activated K-Cl cotransport and shrinkage-activated Na/H exchange. Cells were prepared with intracellular salt concentration and weight percentage of cell water (%cw) varied independently by transient permeabilization of the cell membrane to cations. The dependence of transporter-mediated Na and K influxes upon %cw and upon extracellular salt concentration (c(ext)) was measured in cells so prepared. It was found that the critical value of %cw at which transporters are activated, called the set point, is similar for the two transporters, and that the set points for the two transporters decrease similarly with increasing extracellular salt concentration. These findings suggest a common mechanism of regulation of these two transporters. Cellular Na, K, and Cl concentrations were measured as functions of %cw and c(ext). Using these data together with data from the literature for other solute concentrations, empirical expressions were developed to describe the dependence of the intracellular concentrations of all significant small molecule electrolytes, and therefore the intracellular ionic strength, upon %cw and c(ext). A mechanistic model for the dependence of the set point of an individual transporter upon intracellular ionic strength is proposed. According to this model, the set point represents a critical extent of association between the transporter and a postulated soluble regulatory protein, called regulator. Model functions are presented for the calculation of the thermodynamic activity of regulator, and hence extent of regulator-transporter association, as a function of total intracellular protein concentration (or %cw) and ionic strength. The experimentally observed dependence of set point %cw on c(ext) are simulated using these functions and the empirical expressions described above, together with reasonable but not uniquely determined values of model parameters.

Effects of urea on K-Cl cotransport in sheep red blood cells: evidence for two signals of swelling.

The activation proceeds with a delay, like activation by swelling. Swelling of cells in urea activates K uptake further, but with no delay. Inactivation after removal of urea also proceeds without delay. With cotransport partially activated by reducing intracellular Mg concentration ([Mg]i) or with staurosporine, urea did not activate cotransport further. However, swelling activated cotransport further in these two types of cells. In terms of the three-state process for swelling-activation of K-Cl cotransport (P. B. Dunham, J. Klimczak, and P. J. Logue, J. Gen. Physiol. 101: 733-765, 1993), these results indicate that urea activates the first conversion, A-->B, and does so by inhibiting the reverse reaction promoted by a kinase, just as reducing [Mg]i does. Stimulation of cotransport by urea is nearly completely reversed by shrinkage, whereas activation by reducing [Mg]i is reversed only approximately 35%. Therefore urea inhibits the kinase indirectly, like swelling, by reducing macromolecular crowding of cytoplasmic proteins (A. P. Minton, G. C. Coleclasure, and J. C. Parker. Proc. Natl. Acad. Sci. USA 89: 10504-10506, 1992). Since swelling activates cotransport in two ways, one mimicked by urea and one not, there must be two signals of swelling, one a reduction of macromolecular crowding and the other probably a mechanical signal.

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.

Membrane mechanisms and intracellular signalling in cell volume regulation.

Recent work on selected aspects of the cellular and molecular physiology of cell volume regulation is reviewed. First, the physiological significance of the regulation of cell volume is discussed. Membrane transporters involved in cell volume regulation are reviewed, including volume-sensitive K+ and Cl- channels, K+, Cl- and Na+, K+, 2Cl- cotransporters, and the Na+, H+, Cl-, HCO3-, and K+, H+ exchangers. The role of amino acids, particularly taurine, as cellular osmolytes is discussed. Possible mechanisms by which cells sense their volumes, along with the sensors of these signals, are discussed. The signals are mechanical changes in the membrane and changes in macromolecular crowding. Sensors of these signals include stretch-activated channels, the cytoskeleton, and specific membrane or cytoplasmic enzymes. Mechanisms for transduction of the signal from sensors to transporters are reviewed. These include the Ca(2+)-calmodulin system, phospholipases, polyphosphoinositide metabolism, eicosanoid metabolism, and protein kinases and phosphatases. A detailed model is presented for the swelling-initiated signal transduction pathway in Ehrlich ascites tumor cells. Finally, the coordinated control of volume-regulatory transport processes and changes in the expression of organic osmolyte transporters with long-term adaptation to osmotic stress are reviewed briefly.

Characterization of the proteins of the intestinal Na(+)-K(+)-2Cl- cotransporter.

Absorptive intestinal epithelia, such as that of the winter flounder, absorb salt via a bumetanide-sensitive Na(+)-K(+)-2Cl- cotransport mechanism on the brush-border membrane (BBM). The present study demonstrates the first molecular characterization of the intestinal Na(+)-K(+)-2Cl- cotransporter and its unique regulation. The photoaffinity bumetanide analogue, 4-[3H]benzoyl-5-sulfamoyl-3- (3-thenyloxy)benzoic acid, specifically labeled three groups of proteins in flounder intestinal microsomal membranes (MM): a approximately 180-kDa peptide, prominently labeled, and diffuse bands at approximately 110-70 and 50 kDa, less intensely labeled. Subcellular fractionation revealed a single prominently labeled protein of approximately 170 kDa in BBM but not in basolateral membranes (BLM) and little or no labeling of proteins of approximately 110-70 or 50 kDa. Polyclonal antiserum raised against the Ehrlich ascites cell cotransporter identified a 180-kDa peptide in MM and a 175-kDa peptide (pI approximately 5.4) in BBM but none in BLM or in the cytosol of flounder intestine. As predicted from the regulation of cotransport in this tissue, phosphorylation of this protein is increased by guanosine 3',5'-cyclic monophosphate (cGMP)-dependent but not by adenosine 3',5'-cyclic monophosphate-dependent protein kinase. In addition, phosphorylation of the protein is not increased by protein kinase C or Ca2+/calmodulin-dependent protein kinase but is increased by the phosphatase inhibitor calyculin A. Finally, calyculin A preserves the inhibitory effect of cGMP on ion transport, even in the absence of the nucleotide, suggesting that phosphorylation-dephosphorylation mechanisms are crucial in cotransporter regulation. Thus the flounder intestinal cotransporter is a approximately 175-kDa BBM protein that can be regulated by phosphorylation.

Decline in number of Na-K pumps on low-K+ sheep reticulocytes during maturation is modulated by Lp antigen.

The number of the Na-K pumps on sheep red blood cells declines markedly during cell maturation. In addition, in red blood cells of the low-K+ (LK) phenotype, there is an increase during maturation in the affinity of the pumps for intracellular K+. This increase does not occur in cells of the high-K+ (HK) phenotype. This HK/LK polymorphism is associated with the M/L blood group antigen system. The Lp antigen, which is on only LK cells, promotes the increase in affinity for K+ [Am. J. Physiol. 265 (Cell Physiol. 34): C99-C105, 1993]. Mature LK cells have fewer pumps than mature HK cells. The present study shows that the Lp antigen also promotes the loss of pumps in LK cells. The evidence was that modification of the Lp antigen of immature LK red blood cells either with anti-Lp antibody or by trypsinization diminished the loss of pumps during culture in vitro (numbers determined from [3H]ouabain binding). Confirmation came from demonstration of the decline during maturation of the amount of the alpha-subunit of the Na-K pump (measured by immunoblotting), which was also retarded by pretreatment with anti-Lp or trypsin. Comparisons of the relative amounts of Lp antigen on immature and mature LK cells showed that there is little decline in number of antigens during maturation. Therefore there is an increase in the antigen-to-pump ratio during maturation even though an association between pumps and antigens is necessary for the loss of pumps.

Staurosporine, a protein kinase inhibitor, activates K-Cl cotransport in LK sheep erythrocytes.

K-Cl cotransport can participate in volume regulation in a number of cell types. Swelling activation of K-Cl cotransport in sheep erythrocytes proceeds by a two-step process, A<-->B<-->C (Dunham et al., J. Gen. Physiol. 101: 733-765, 1993). The A state, with a low flux, predominates at physiological volume. A-->B is rate limiting and can be activated by reducing cell Mg concentration ([Mg]c); complete activation (B-->C) requires cell swelling. Inhibitors of protein kinases and phosphatases were employed in an attempt to identify enzymatic reactions in the activation process. Staurosporine, a kinase inhibitor, activated K-Cl cotransport by approximately sixfold. Swelling of staurosporine-treated cells caused further activation that proceeded without delay. The effects of staurosporine and reducing [Mg]c were not additive. These two results indicate that staurosporine, like reducing [Mg]c, promotes the rate-limiting A-->B conversion. Unlike swelling, staurosporine activated cotransport without delay. Therefore staurosporine activates by promoting the forward reaction in the A<-->B conversions, in contrast to swelling, which activates by inhibiting the reverse reaction. Calyculin A, a phosphatase inhibitor, inhibited K-Cl cotransport but did not inhibit after activation by reducing [Mg]c, confirming earlier proposals that A-->B is promoted by a phosphatase. Calyculin A, added before or after staurosporine, abolished activation by staurosporine, confirming that staurosporine promotes A-->B. It is proposed that the phosphatase promoting this reaction is regulated by an inhibitory kinase, the staurosporine target.

The Na-K-2Cl cotransporter is in a permanently activated state in cytoplasts from Ehrlich ascites tumor cells.

Brief incubation of Ehrlich ascites tumor cells with cytochalasin B causes the formation of blebs in the surface membrane. Gentle homogenization removes the blebs as intact cytoplasts which contain neither mitochondrian or nucleus, nor other cytoplasmic membranous organelles. The Na-K-2Cl cotransporter is present in the cytoplasts in a permanently activated state, whereas the Na-K-2Cl transport system in unperturbed intact cells is silent. Pretreatment of intact cells with cytochalasin B for 1 min stimulates the bumetanide-inhibitable K+ influx approximately fivefold. The influx into purified cytoplasts when expressed per g protein is three- to fourfold higher than the influx into cytochalasin B-treated intact cells. Thus, the membrane vesicles are enriched with the cotransporter, and the cotransporter is present in an activated state. The K influx into cytoplasts is inhibited about 40% by Na-free, Cl-free or bumetanide-containing media and to a similar extent by Fab fragments prepared from antiserum against purified proteins of the cotransporter. The KI for bumetanide was 0.19 +/- 0.06 microM for the cytoplasts as compared to 0.67 +/- 0.11 microM for the intact cells. SDS gel electrophoresis of membrane proteins from the cytoplast membranes compared to the membranes of intact cells shows a reduced number of bands and a majority of bands showing reduced staining, whereas a few bands are stained more intensely. Particularly notable is a band at approximately 80 kD, which is similar to the molecular weight previously reported for the main membrane protein isolated from intact cells using a bumetanide-Sepharose affinity column. An immunoblot of the cytoplast preparation using antibodies against the purified bumetanide binding proteins showed strong immunodetection of the approximately 80 kD protein.

Characterization of the endogenous Na(+)-K(+)-2Cl- cotransporter in Xenopus oocytes.

Over time, Xenopus laevis changed from producing stage V and VI oocytes with little native Na(+)-K(+)-2Cl- cotransport activity to those with substantial activity. In oocytes with high endogenous activity, K+ uptake, using the tracer 86Rb+ was approximately 20 pmol.min-1.oocyte-1 in the presence of blockers of Na(+)-K(+)-ATPase and conductive K+ transport. Bumetanide (10 microM) inhibited > 90% of this uptake, suggesting involvement of Na(+)-K(+)-2Cl- cotransport. This was confirmed by two observations that are found in this cotransporter in other tissues: 1) The related diuretics, thiobenzmetanide [50% inhibitory concentration (IC50), 2 x 10(-11) M] > bumetanide (IC50, 7 x 10(-8) M) > furosemide (IC50, 2.5 x 10(-6) M) inhibited the cotransporter in a dose-dependent manner. 2) There was little uptake of K+ in the absence of extracellular Na+ or Cl-. Halving medium osmolarity to 92 mosM decreased bumetanide-sensitive K+ uptake by approximately 75%, whereas a doubling of medium osmolarity increased it by approximately 50%. The cotransport activity was increased fourfold by the phosphatase inhibitor calyculin A (200 nM) but was unaffected by 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate, 8-bromoguanosine 3',5'-cyclic monophosphate, ATP, ionomycin, or okadaic acid. Both the photoaffinity bumetanide analogue, 4-[3H]benzoyl-5-sulfamoyl-3-(3-thenyloxy)benzoic acid, and an antiserum raised against Ehrlich ascites cell cotransporter specifically labeled an approximately 140-kDa oocyte membrane protein. These results demonstrated that, in addition to the Na+ pump and K+ channels, K+ uptake in Xenopus oocytes occurs via a loop-diuretic-sensitive Na(+)-K(+)-2Cl- cotransporter.(ABSTRACT TRUNCATED AT 250 WORDS)