Identification and reconstitution of a Na/K/Cl cotransporter and K channel from luminal membranes of renal red outer medulla

Electrophysiological studies on renal thick ascending limb segments indicate the involvement of a luminal Na⁺/K⁺/Cl⁻ cotransport system and a K⁺ channel in transepithelial salt transport. Sodium reabsorption across this segment is blocked by the diuretics furosemide and bumetanide. The object of our study has been to identify in intact membranes and reconstitute into phospholipid vesicles the Na⁺/K⁺/Cl⁻ cotransporter and K⁺ channel, as an essential first step towards purification of the proteins involved and characterization of their roles in the regulation of transepithelial salt transport. Measurements of ⁸⁶Rb⁺ uptake into membrane vesicles against large opposing KCl gradients greatly magnify the ratio of specific compared to non-specific isotope flux pathways. Using this sensitive procedure, it has proved possible to demonstrate in crude microsomal vesicle preparations from rabbit renal outer medulla two ⁸⁶Rb⁺ fluxes. (A) A furosemide-inhibited ⁸⁶Rb⁺ flux in the absence of Na⁺ (K⁺-K⁺ exchange). This flux is stimulated by an inward Na⁺ gradient (Na⁺/K⁺ cotransport) and is inhibited also by bumetanide. (B) A Ba²⁺-inhibited ⁸⁶Rb⁺ flux, through the K⁺ channel. Luminal membranes containing the Na⁺/K⁺/Cl⁻ cotransporter and K⁺ channels, and basolateral membranes containing the Na⁺/K⁺ pumps were separated from the bulk of contaminant protein by metrizamide density gradient centrifugation. The Na⁺/K⁺/Cl⁻ cotransporter and K⁺ channel were reconstituted in a functional state by solubilizing both luminal membranes and soybean phospholipid with octyl glucoside, and then removing detergent on a Sephadex column.     

Growth hormone and cortisol treatment stimulate seawater tolerance in both anadromous and landlocked Arctic charr

In a comparative experiment the effect of cortisol and growth hormone (GH) on the hypo-osmoregulatory ability of a landlocked and an anadromous strain of Arctic charr (Salvelinus alpinus) was investigated. Cortisol and GH were implanted either alone or in combination, and the fish were exposed to a 24 h seawater challenge test (SWT) on days 14 and 28 after implantation. Hypo-osmoregulatory ability, measured as plasma osmolality and chloride concentration after the SWTs, was better in the anadromous than in the landlocked strain, irrespective of treatment. However, cortisol provided a strong stimulation of hypo-osmoregualtory ability in both strains, and this stimulation seemed to be potentiated by GH in an additive manner. Improved hypo-osmoregulatory ability in GH + cortisol treated anadromous Arctic charr was accompanied by increased gill Na⁺, K⁺-ATPase activity and Na⁺–K⁺–2Cl⁻ cotransporter protein abundance, but no changes in gill Na⁺,K⁺-ATPase α1a and α1b mRNA levels. For landlocked charr the improved hypo-osmoregulatory ability in GH +cortisol treated fish was accompanied only with an increase in gill Na⁺–K⁺–2Cl⁻ cotransporter protein abundance. Hormone treatment caused an improvement of hypo-osmoregulatory ability that was of approximately the same magnitude in the landlocked as in the anadromous Arctic charr. This suggests that the lack of spontaneous development of hypo-osmoregulatory ability often seen in landlocked populations of Arctic charr may depend, at least partly, on a lack of the hormonal activation seen in anadromous populations.     

Simulation analysis of intracellular Na and Cl homeostasis during β1-adrenergic stimulation of cardiac myocyte

To quantitatively understand intracellular Na⁺ and Cl⁻ homeostasis as well as roles of Na⁺/K⁺ pump and cystic fibrosis transmembrane conductance regulator Cl⁻ channel (I_CFTR) during the β1-adrenergic stimulation in cardiac myocyte, we constructed a computer model of β1-adrenergic signaling and implemented it into an excitation-contraction coupling model of the guinea-pig ventricular cell, which can reproduce membrane excitation, intracellular ion changes (Na⁺, K⁺, Ca²⁺ and Cl⁻), contraction, cell volume, and oxidative phosphorylation. An application of isoproterenol to the model cell resulted in the shortening of action potential duration (APD) after a transient prolongation, the increases in both Ca²⁺ transient and cell shortening, and the decreases in both Cl⁻ concentration and cell volume. These results are consistent with experimental data. Increasing the density of I_CFTR shortened APD and augmented the peak amplitudes of the L-type Ca²⁺ current (I_CaL) and the Ca²⁺ transient during the β1-adrenergic stimulation. This indirect inotropic effect was elucidated by the increase in the driving force of I_CaL via a decrease in plateau potential. Our model reproduced the experimental data demonstrating the decrease in intracellular Na⁺ during the β-adrenergic stimulation at 0 or 0.5 Hz electrical stimulation. The decrease is attributable to the increase in Na⁺ affinity of Na⁺/K⁺ pump by protein kinase A. However it was predicted that Na⁺ increases at higher beating rate because of larger Na⁺ influx through forward Na⁺/Ca²⁺ exchange. It was demonstrated that dynamic changes in Na⁺ and Cl⁻ fluxes remarkably affect the inotropic action of isoproterenol in the ventricular myocytes.     

Measurement and stoichiometry of bumetanide-sensitive (2Na∶1K∶3Cl) cotransport in ferret red cells

Summary The bumetanide-sensitive uptake of Na⁺, K^–(Rb^–) and Cl^– has been measured at 21°C in ferrent red cells treated with (SITS+DIDS) to minimize anion flux via capnophorin (Band 3). During the time course of the influx experiments tracer uptake was a first-order rate process. At normal levels of external Na⁺ (150mm) the bumetanide-sensitive uptake of K⁺ was dependent on Cl^– and represented almost all of the K⁺ uptake, the residual flux demonstrating linear concentration dependence. The uptake of Na⁺ and Cl^– was only partially inhibited by bumetanide indicating that pathways other than (Na+K+Cl) cotransport participate in these fluxes. The diuretic-sensitive uptake of Na⁺ or Cl^– was, however, abolished by the removal of K⁺ or the complementary ion indicating that bumetanide-sensitive fluxes of Na⁺, K⁺ and Cl^– are closely coupled. At very low levels of [Na] _o (<5mm) K⁺ influx demonstrated complex kinetics, and there was evidence of the unmasking of a bumetanide-sensitive Na⁺-independent K⁺ transport pathway. The stoichiometry of bumetanide-sensitive tracer uptake was 2Na1K3Cl both in cells suspended in a low and a high K⁺-containing medium. The bumetanide-sensitive flux was markedly reduced by ATP depletion. We conclude that a bumetanide-sensitive cotransport of (2Na1K3Cl) occurs as an electroneutral complex across the ferret red cell membrane.     

Na+, K+, Cl− cotransport and its regulation in Ehrlich ascites Tumor cells. Ca2+/Calmodulin and protein kinase C dependent pathways

Summary Net Cl^– uptake as well as unidirectional³⁶Cl influx during regulatory volume increase (RVI) require external K⁺. Half-maximal rate of bumetanide-sensitive³⁶Cl uptake is attained at about 3.3mm external K⁺. The bumetanide-sensitive K⁺ influx found during RVI is strongly dependent on both Na⁺ and Cl^–. The bumetanide-sensitive unidirectional Na⁺ influx during RVI is dependent on K⁺ as well as on Cl^–. The cotransporter activated during RVI in Ehrlich cells, therefore, seems to transport Na⁺, K⁺ and Cl^–. In the presence of ouabain and Ba⁺ the stoichiometry of the bumetanide-sensitive net fluxes can be measured at 1.0 Na⁺, 0.8 K⁺, 2.0 Cl^– or approximately 1 : Na, 1 : K, 2 : Cl. Under these circumstances the K⁺ and Cl^– flux ratios (influx/efflux) for the bumetanide-sensitive component were estimated at 1.34 ±0.08 and 1.82 ± 0.15 which should be compared to the gradient for the Na⁺, K⁺, 2Cl^– cotransport system at 1.75 ± 0.24.Addition of sucrose to hypertonicity causes the Ehrlich cells to shrink with no signs of RVI, whereas shrinkage with hypertonic standard medium (all extracellular ion concentrations increased) results in a RVI response towards the original cell volume. Under both conditions a bumetanide-sensitive unidirectional K⁺ influx is activated. During hypotonic conditions a small bumetanide-sensitive K⁺ influx is observed, indicating that the cotransport system is already activated.The cotransport is activated 10–15 fold by bradykinin, an agonist which stimulates phospholipase C resulting in release of internal Ca²⁺ and activation of protein kinase C.The anti-calmodulin drug pimozide inhibits most of the bumetanide-sensitive K⁺ influx during RVI. The cotransporter can be activated by the phorbol ester TPA. These results indicate that the stimulation of the Na⁺, K⁺, Cl^– cotransport involves both Ca²⁺/calmodulin and protein kinase C.     

Vasopressin alters the mechanism of apical Cl− entry from Na+:Cl− to Na+:K+:2Cl− cotransport in mouse medullary thick ascending limb

Summary Experiments were performed usingin vitro perfused medullary thick ascending limbs of Henle (MTAL) and in suspensions of MTAL tubules isolated from mouse kidney to evaluate the effects of arginine vasopressin (AVP) on the K⁺ dependence of the apical, furosemide-sensitive Na⁺:Cl^– cotransporter and on transport-related oxygen consumption (QO₂). In isolated perfused MTAL segments, the rate of cell swelling induced by removing K⁺ from, and adding onemm ouabain to, the basolateral solution [ouabain(zero-K⁺)] provided an index to apical cotransporter activity and was used to evaluated the ionic requirements of the apical cotransporter in the presence and absence of AVP. In the absence of AVP cotransporter activity required Na⁺ and Cl^–, but not K⁺, while in the presence of AVP the apical cotransporter required all three ions.⁸⁶Rb⁺ uptake into MTAL tubules in suspension was significant only after exposure of tubules to AVP. Moreover,²²Na⁺ uptake was unaffected by extracellular K⁺ in the absence of AVP while after AVP exposure²²Na⁺ uptake was strictly K⁺-dependent. The AVP-induced coupling of K⁺ to the Na⁺:Cl^– cotransporter resulted in a doubling in the rate of NaCl absorption without a parallel increase in the rate of cellular²²Na⁺ uptake or transport-related oxygen consumption. These results indicate that arginine vasopressin alters the mode of a loop diuretic-sensitive transporter from Na⁺:Cl^– cotransport to Na⁺:K⁺:2Cl^– cotransport in the mouse MTAL with the latter providing a distinct metabolic advantage for sodium transport. A model for AVP action on NaCl absorption by the MTAL is presented and the physiological significance of the coupling of K⁺ to the apical Na⁺:Cl^– cotransporter in the MTAL and of the enhanced metabolic efficiency are discussed.     

Transepithelial Taurine Transport in Caco-2 Cell Monolayers

Here we characterized transepithelial taurine transport in monolayers of cultured human intestinal Caco-2 cells by analyzing kinetic apical and basolateral uptake and efflux parameters. Basolateral uptake was Na⁺- and Cl⁻- dependent and was inhibited by β-amino acids. Uptake by this membrane showed properties similar to those of the apical TauT system. In both membranes, taurine uptake fitted a model consisting of a non-saturable plus a saturable component, with a higher half-saturation constant and transport capacity at the apical membrane (K_m, 17.1 μmol/L; V_max, 28.4 pmol·cm⁻²·5 min⁻¹) than in the basolateral domain (K_m, 9.46 μmol/L; V_max, 5.59 pmol·cm⁻²·5 min⁻¹). The non-saturable influx component, estimated in the absence of Na⁺ and Cl⁻, showed no significant differences between apical and basolateral membranes (K_D, 89.2 and 114.7 nL·cm⁻² · 5 min⁻¹, respectively). Taurine efflux from the cells is a diffusive process, as shown in experiments using preloaded cells and in trans-stimulation studies (apical K_D,72.7 and basolateral K_D, 50.1 nL·cm⁻²·5 min⁻¹). Basolateral efflux rates were significantly lower than passive influx rates. We conclude that basolateral taurine uptake in Caco-2 cells is mediated by a transport mechanism that shares some properties with the apical system TauT. Moreover, calculation of unidirectional and transepithelial taurine fluxes reveals that apical influx of this amino acid is higher than basolateral efflux rates, thereby enabling epithelial cells to accumulate taurine against a concentration gradient.     

Gill (Na+,K+)-ATPase from the blue crab Callinectes danae: modulation of K+-phosphatase activity by potassium and ammonium ions

The kinetic properties of a microsomal gill (Na⁺,K⁺)-ATPase from the blue crab Callinectes danae were analyzed using the substrate p-nitrophenylphosphate. The (Na⁺,K⁺)-ATPase hydrolyzed PNPP obeying cooperative kinetics (n=1.5) at a rate of V=125.4±7.5 U mg⁻¹ with K_0.5=1.2±0.1 mmol l⁻¹; stimulation by potassium (V=121.0±6.1 U mg⁻¹; K_0.5=2.1±0.1 mmol l⁻¹) and magnesium ions (V=125.3±6.3 U mg⁻¹; K_0.5=1.0±0.1 mmol l⁻¹) was cooperative. Ammonium ions also stimulated the enzyme through site–site interactions (n_H=2.7) to a rate of V=126.1±4.8 U mg⁻¹ with K_0.5=13.7±0.5 mmol l⁻¹. However, K⁺-phosphatase activity was not stimulated further by K⁺ plus NH₄⁺ ions. Sodium ions (K_I=36.7±1.7 mmol l⁻¹), ouabain (K_I=830.3±42.5 μmol l⁻¹) and orthovanadate (K_I=34.0±1.4 nmol l⁻¹) completely inhibited K⁺-phosphatase activity. The competitive inhibition by ATP (K_I=57.2±2.6 μmol l⁻¹) of PNPPase activity suggests that both substrates are hydrolyzed at the same site on the enzyme. These data reveal that the K⁺-phosphatase activity corresponds strictly to a (Na⁺,K⁺)-ATPase in C. danae gill tissue. This is the first known kinetic characterization of K⁺-phosphatase activity in the portunid crab C. danae and should provide a useful tool for comparative studies.     

Relation between Ion Accumulation of Salt-Sensitive and Isolated Stable Salt-Tolerant Cell Lines of Citrus aurantium

Four selected NaCl-tolerant cell lines of Sour orange (Citrus aurantium) were compared with the nonselected cell line in their growth and internal ion content of Na⁺, K⁺, and Cl⁻ when exposed to increasing NaCl concentrations. No difference was found among the various NaCl-tolerant cell lines in Na⁺ and Cl⁻ uptake, and all these cell lines took up similar or even larger amounts of Na⁺ and Cl⁻ than the NaCl-sensitive cell line. Exposure of cells of NaCl-sensitive and NaCl-tolerant lines to equal external concentrations of NaCl, resulted in a greater loss of K⁺ from the NaCl-sensitive cell line. This observation leads to the conclusion that growth and ability to retain high levels of internal K⁺ are correlated. Exposure of the NaCl-tolerant cell lines to salts other than NaCl resulted in even greater tolerance to Na₂SO₄, but rather poor tolerance to K⁺ introduced as either K₂SO₄ or KCl; the latter has a stronger inhibitory effect. The NaCl-sensitive cell line proved to be more sensitive to replacement of Na⁺ by K⁺. Analyses of internal Na⁺, K⁺, and Cl⁻ concentrations failed to identify any particular internal ion concentration which could serve as a reliable marker for salt tolerance.     

Driving forces in hepatocellular uptake of phalloidin and cholate

Active uptake of phalloidin and cholate in isolated rat liver cells depends upon both Na⁺ gradient and membrane potential. Omission of Na⁺ or inhibition of the (Na⁺ + K⁺)-ATPase diminished both phalloidin and cholate uptake. Dissipation of the sodium, potassium or proton gradient by monensin, nigericin, gramicidin and valinomycin blocked phalloidin uptake and also caused reduction of cholate transport. Chelation of Ca²⁺ and Mg²⁺ by EGTA or incubation of liver cells with NH₄Cl neither influenced phalloidin nor cholate uptake. Hyperpolarization of liver cells by the lipophilic anions NO₃⁻ or SCN⁻ enhanced phalloidin but reduced cholate uptake. Depolarization induced by a reversed K⁺ gradient reduced both kinds of transport. The results indicate that sodium ions and the membrane potential are driving forces for phalloidin and cholate uptake in hepatocytes.     

Freshwater to seawater acclimation of juvenile bull sharks (<Emphasis Type="Italic">Carcharhinus leucas</Emphasis>): plasma osmolytes and Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase activity in gill,rectal gland,kidney and intestine

This study examined the osmoregulatory status of the euryhaline elasmobranch Carcharhinus leucas acclimated to freshwater (FW) and seawater (SW). Juvenile C. leucas captured in FW (3 mOsm l^–1 kg^–1) were acclimated to SW (980–1,000 mOsm l^–1 kg^–1) over 16 days. A FW group was maintained in captivity over a similar time period. In FW, bull sharks were hyper-osmotic regulators, having a plasma osmolarity of 595 mOsm l^–1 kg^–1. In SW, bull sharks had significantly higher plasma osmolarities (940 mOsm l^–1 kg^–1) than FW-acclimated animals and were slightly hypo-osmotic to the environment. Plasma Na⁺, Cl^–, K⁺, Mg²⁺, Ca²⁺, urea and trimethylamine oxide (TMAO) concentrations were all significantly higher in bull sharks acclimated to SW, with urea and TMAO showing the greatest increase. Gill, rectal gland, kidney and intestinal tissue were taken from animals acclimated to FW and SW and analysed for maximal Na⁺/K⁺-ATPase activity. Na⁺/K⁺-ATPase activity in the gills and intestine was less than 1 mmol Pi mg^–1 protein h^–1 and there was no difference in activity between FW- and SW-acclimated animals. In contrast Na⁺/K⁺-ATPase activity in the rectal gland and kidney were significantly higher than gill and intestine and showed significant differences between the FW- and SW-acclimated groups. In FW and SW, rectal gland Na⁺/K⁺-ATPase activity was 5.6±0.8 and 9.2±0.6 mmol Pi mg^–1 protein h^–1, respectively. Na⁺/K⁺-ATPase activity in the kidney of FW and SW acclimated animals was 8.4±1.1 and 3.3±1.1 Pi mg^–1 protein h^–1, respectively. Thus juvenile bull sharks have the osmoregulatory plasticity to acclimate to SW; their preference for the upper reaches of rivers where salinity is low is therefore likely to be for predator avoidance and/or increased food abundance rather than because of a physiological constraint.     

Relationship between coupled Na+−K+−Cl− transport and water absorption across the seawater eel intestine

Summary Simultaneous measurements of net ion and water fluxes were made in the stripped intestine of the seawater eel, and the relationship between Na⁺, K⁺, Cl^– and water transport were examined in the presence of mucosal KCl and serosal NaCl Ringer (standard condition). When Cl^– was removed from both sides of the intestine, net K⁺ flux from mucosa to serosa was reduced, accompanied by complete blockage of water absorption. Since it has been shown that net Cl^– and water fluxes depend on K⁺ transport under the standard condition (Ando 1983), the interdependence of K⁺ and Cl^– transport suggests the existence of a coupled KCl transport system, while the parallelism between the net Cl^– and water fluxes suggests that water absorption is linked to the coupled KCl transport. The coupled KCl and water transport were inhibited by treatment with ouabain or with Na⁺-free Ringer solutions, suggesting the existence of a Na⁺-dependent KCl transport system and linkage of water absorption to the coupled Na⁺–K⁺–Cl^– transport. Since ouabain blocked the active Na⁺–K⁺–Cl^– transport almost completely, the permeability coefficients for K⁺ and Na⁺ through the paracellular shunt pathway were estimated as P_K=0.076 and P_Na=0.058 cm/h, and P_Cl was calculated as 0.005 cm/h. Although Na⁺-independent K⁺ and Cl^tt- fluxes were observed again in the present study, these fluxes were not inhibited by CN^–, ouabain or diuretics, and evoked even after blocking the Na⁺–K⁺–Cl^– transport completely with ouabain. These results indicate that the Na⁺-independent K⁺ and Cl^– fluxes are distinct from the active Na⁺–K⁺–Cl^– transport and are not themselves active.     

A reconstituted Na++K+ pump in liposomes containing purified (Na++K+-ATPase from kidney medulla

Liposomes containing either purified or microsomal (Na⁺,K⁺)-ATPase preparations from lamb kidney medulla catalyzed ATP-dependent transport of Na⁺ and K⁺ with a ratio of approximately 3Na⁺ to 2K⁺, which was inhibited by ouabain. Similar results were obtained with liposomes containing a partially purified (Na⁺,K⁺-ATPase from cardiac muscle. This contrasts with an earlier report by Goldin and Tong (J. Biol. Chem. 249, 5907–5915, 1974), in which liposomes containing purified dog kidney (Na⁺,K⁺)-ATPase did not transport K⁺ but catalyzed ATP-dependent symport of Na⁺ and Cl^?. When purified by our procedure, dog kidney (Na⁺,K⁺)-ATPase showed some ability to transport K⁺ but the ratio of Na⁺ : K⁺ was 5 : 1.     

Calculations of K+, Na+, and Cl? fluxes across cell membrane with Na+/K+ pump, NKCC, NC cotransport and ionic channels with non-Goldman rectification in K+-channels: Normal and apoptotic cells

The balance of K⁺, Na⁺, and Cl⁻ fluxes across the cell membrane with the Na⁺/K⁺ pump, ion channels, and Na⁺K⁺2Cl⁻ (NKCC) and Na⁺-Cl⁻ (NC) cotransport was calculated to determine the mechanism of cell shrinkage in apoptosis. It is shown that all unidirectional K⁺, Na⁺, and Cl⁻ fluxes; the ion channel permeability; and the membrane potential can be found using the principle of the flux balance if the following experimental data are known: K⁺, Na⁺, and Cl⁻ concentrations in cell water; total Cl⁻ flux; total K⁺ influx; and the ouabain-inhibited pump component of the Rb⁺(K⁺) influx. The change in different ionic pathways during apoptosis was estimated by calculations based on the data reported in the preceded paper (Yurinskaya et al., 2010). It is found that cell shrinkage and the shift in ion balance in U937 cells induced to apoptosis with 1 μM staurosporine occur due to the coupling of reduced pump activity with a decrease in the integral permeability of Na⁺ channels, whereas K⁺ and Cl⁻ channel permeability remains almost unchanged. Calculations show that only a small part of the total fluxes of K⁺, Na⁺, and Cl⁻ account for the fluxes mediated by NKCC and NC cotransporters. Despite the importance of cotransport fluxes for maintaining the nonequilibrium steady-state distribution of Cl⁻, they cannot play a significant role in apoptotic cell shrinkage because of their minority and cannot be revealed by inhibitors.     

A comparative study on the effects of different bile salts on mucosal ATPase and transport in the rat jejunum in vivo

The effects of deoxycholate, taurocholate and cholate on transport and mucosal ATPase activity have been investigated in the rat jejunum in vivo using closed-loop and perfusion techniques.In the closed-loops, 5 mM deoxycholate selectively inactivated (Na⁺ + K⁺)-ATPase, and net secretion of Na⁺ induced by 2.5 mM deoxycholate was due to reduced lumen to plasma flux of the ion; deoxycholate (2.5 mM) produced marked inhibition of 3-O-methylglucose transport. Luminal disappearance rates of deoxycholate (60.5±2.9 % per g wet wt of gut) greatly exceeded those of taurocholate (4.3±1.0).In the perfusion studies 1 mM deoxycholate induced net secretion of water, Na⁺ and Cl⁻, and inhibited active glucose transport; concomitantly “total” ATPase, (Na⁺ + K⁺)-ATPase, and Mg²⁺-ATPase were inhibited. At higher concentrations (5 mM) deoxycholate stimulated Mg²⁺-ATPase activity. Taurocholate and cholate at 1 mM had no effect on transport or (Na⁺ + K⁺)-ATPase. Mucosal lactase, sucrase and maltase activities were not affected by 1 mM deoxycholate, taurocholate or cholate.These results suggest that deoxycholate inhibits sodium-coupled glucose transport by inhibition of (Na⁺ + K⁺)-ATPase at the lateral and basal membranes of the epithelial cell, rather than from an effect at the brush-border membrane level.     

Conformational changes of membrane-bound (Na+−K+)-ATPase as revealed by trypsin digestion

Summary To distinguish ligand-induced structural states of the (Na⁺–K⁺)-ATPase, the purified membrane-bound enzyme isolated from rat kidneys was digested with trypsin in the presence of various combinations of Na⁺, K⁺, Mg⁺⁺ and ATP. It was found that first the large and then the small polypeptide chain of the (Na⁺–K⁺)-ATPase was degraded, indicating that the lysine and arginine residues of the large chain are more exposed than are those of the small one. The (Na⁺–K⁺)-ATPase activity was inactivated in parallel with the degradation of the large polypeptide chain. After the degradation of the large polypeptide chain, about 75% of the (Na⁺–K⁺)-ATPase protein remained bound to the membrane, demonstrating that the split protein segments were only partially released.It was found that the combinations of ATP, Mg⁺⁺, Na⁺ and K⁺ present during trypsin digestion influenced the time course and degree of degradation of the (Na⁺–K⁺)-ATPase protein. The degradations of the large and the small polypeptide chain were affected in parallel. Thus, certain ATP and ligand combinations influenced neither the degradation of the large nor the degradation of the small polypeptide chain, whereas by other combinations of ATP and ligands the degree of susceptibility of both polypeptide chains to trypsin was equally increased or reduced.In the absence of ATP the time course of trypsin digestion of the (Na⁺–K⁺)-ATPase was the same, whether Na⁺ or K⁺ was present. With low ATP concentrations (e.g., 0.1mm), however, binding of Na⁺ or K⁺ led to different degradation patterns of the enzyme. If a high concentration of ATP (e.g., 10mm) was present, Na⁺ and K⁺ also influenced the degradation pattern of the (Na⁺–K⁺)-ATPase, but differentially compared to that at low ATP concentrations, since the effects of Na⁺ and K⁺ were reversed. Furthermore, it was found that the degradation of the small chain was only influenced by certain combinations of ATP, Mg⁺⁺, Na⁺ and K⁺ if the large chain was intact when the ligands were added to the enzyme.The described results demonstrate structural alterations of the (Na⁺–K⁺)-ATPase complex which are supposed to include a synchronous protrusion or retraction of both (Na⁺–K⁺)-ATPase subunits. The data further suggest that ATP and other ligands primarily alter the structure of the large (Na⁺–K⁺)-ATPase subunit. This structural alteration is presumed to lead to a synchronous movement of the small subunit of the enzyme. The structural state of the (Na⁺–K⁺)-ATPase is regulated by binding of Na⁺ or K⁺ to the enzyme-ATP complex. The effects of Na⁺ and K⁺ on the (Na⁺–K⁺)-ATPase structure are modulated by the ATP binding to high affinity and to low affinity ATP binding sites.     

Sidium-dependent ion cotransport in steady-state Ehrlich ascites tumor cells

Summary The Ehrlich tumor cell possesses and anion-cation cotransport system which operates as a bidirectional exchanger during the physiological steady state. This cotransport system, like that associated with the volume regulatory mechanism (i.e. coupled net uptake of Cl^–+Na⁺ and/or K⁺) is Cl^–-selective and furosemide-sensitive, suggesting the same mechanism operating in two different modes. Since Na⁺ has an important function in the volume regulatory response, its role in steady-state cotransport was investigated. In the absence of Na⁺, ouabain-insensitive K⁺ and DIDS-insensitive Cl^– transport (KCl cotransport) are low and equivalent to that found in 150mm Na⁺ medium containing furosemide. Increasing the [Na⁺] results in parallel increases in K⁺ and Cl^– transport. The maximum rate of each (18 to 20 meq/(kg dry wt)·min) is reached at about 20mm Na⁺ and is maintained up to 55mm. Thus, over the range 1 to 55mm Na⁺ the stoichiometry of KCl cotransport is 11. In contrast to K⁺ and Cl^–, furosemide-sensitive Na⁺ transport is undetectable until the [Na⁺] exceeds 50mm. From 50 to 150mm Na⁺, it progressively rises to 7 meq/(kg dry wt)·min, while K⁺ and Cl^– transport decrease to 9 and 16 meq/(kg dry wt)·min, respectively. Thus, at 150mm Na⁺ the stoichiometric relationship between Cl^–, Na⁺ and K⁺ is 211. These results are consistent with the proposal that the Cl^–-dependent cation cotransport system when operating during the steady state mediates the exchange of KCl for KCl or NaCl for NaCl; the relative proportion of each determined by the extracellular [Na⁺].