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We report here our studies on K+ transport in resealed human red cell ghosts (RG) in the presence of 0.1 mM ouabain and 0.01 mM bumetanide, inhibitors of the Na+-K+ pump and Na+-K+-Cl- cotransport, respectively. RG were obtained with the gel-filtration method. K+ efflux from RG was dependent on the pH used in the lysis buffer and increased when the pH used in the lysis buffer and increased when the pH was raised from 5.5 to 8.0. As in intact red cells, RG made from cells of the least dense fraction had a much higher K+ efflux than RG made from cells of the densest fraction. This K+ flux is volume independent and increases when the pH of the flux medium is increased from 6.0 to 8.0. K+ efflux (60-70%) at pH 7.40 from RG made from cells of the least dense fraction is inhibited when Cl- is substituted by nitrate or when the ghosts are resealed in the absence of ATP. This chloride- and ATP-dependent component is markedly reduced in RG made from cells of the densest fraction. An increase in the internal Mg2+ concentration in RG from the least dense fraction induced marked inhibition of K+ efflux. Contrary to intact cells, N-ethylmaleimide (NEM) did not affect K+ efflux from RG. Thus the effects of pH, osmolarity, and NEM on K+ transport in RG are markedly different from those reported in intact erythrocytes.

In this paper, we report experiments demonstrating the coupling of Cl and K movements in a volume-dependent K-Cl cotransport system in human red blood cells. We show that an outwardly directed Cl gradient can promote net K efflux against an inwardly directed K gradient at constant membrane potential. Red blood cell membrane potential was kept constant by using anions that are not transported through the K-Cl cotransport system but that are more permeable than Cl (NO3 and SCN). Under these conditions, when the activities of band 3 (capnophorin)-mediated anion exchange and of the carbonic anhydrase have been inhibited, it is possible to maintain a Cl gradient at constant membrane potential. Similar data were obtained in human red blood cells (least dense fraction from normal subjects and whole blood from patients with homozygous hemoglobin S disease), in rabbit red blood cells, and in low-K sheep red blood cells. These data confirm that the volume-dependent Cl-dependent K movement in these cells operates through coupled K-Cl cotransport.

We report here studies on the regulation of cell volume and K transport in human erythrocytes separated according to density. When cell volume was increased (isosmotic swelling, nystatin technique), erythrocytes of the least dense but not of the densest fraction shrunk back toward their original volume. This process was due to a ouabain (0.1 mM) and bumetanide (0.01 mM) (OB)-resistant K loss. OB-resistant K+ efflux from the least dense fraction was stimulated by hypotonic swelling and had a bell-shaped dependence on pH (pH optimum 6.75–7.0). These pH and volume effects were not evident in the densest fraction. The swelling-induced K+ efflux from the least dense fraction was inhibited when chloride was substituted by nitrate, thiocyanate, and acetate, whereas it was stimulated by bromide. Increasing cell Mg2+ content also markedly inhibited K+ efflux from isosmotically swollen cells. N-ethylmaleimide (NEM, 1 mM) greatly increased OB-resistant K+ efflux from the least dense fraction but not from the densest fraction. These data reveal the presence, in the lease dense fraction of normal human erythrocytes, of a pathway for K+ transport that is dependent on volume, pH, and chloride, is inhibited by internal Mg2+, and possibly plays a role in determining the erythrocyte water and cation content.

The Na-channel polypeptide is responsible for the voltage-gated and time-dependent ionic permeability changes that give rise to the action potential in the membranes of nerve cells. We have synthesized a 22-amino acid peptide with a sequence identical to that of the segment named S4, repeat IV of the primary structure of the Na channel. We have found that this peptide induces a voltage- and time-dependent conductance in bilayers formed by a mixture of phosphatidyl-ethanolamine and phosphatidylserine. This conductance is activated when the cis side is made positive, with an apparent gating charge of 3. The results are consistent with the idea that this segment plays a role in determining the voltage sensitivity of the Na channel.

This paper reports experiments designed to find the concentrations of internal and external Na and K at which inward and outward furosemide-sensitive (FS) Na and K fluxes are equal, so that there is no net FS movement of Na and K. The red cell cation content was modified by using the ionophore nystatin, varying cell Na (Nai) from 0 to 34 mM (K substitution, high-K cells) and cell K (Ki) from 0 to 30 mM (Na substitution, high-Na cells). All incubation media contained NaCl (Nao = 130 or 120 nM), and KCl (Ko = 0-30 mM). In high-K cells, incubated in the absence of Ko, there was net extrusion of Na through the FS pathway. The net FS Na extrusion increased when Nai was increased. Low concentrations of Ko (0-6 mM) slightly stimulated, whereas higher concentrations of Ko inhibited, FS Na efflux. Increasing Ko stimulated the FS Na influx (K0.5 = 4 mM). Under conditions similar to those that occur in vivo (Nai = 10, Ki = 130, Nao = 130, Ko = 4 mM, Cli/Clo = 0.7), net extrusion of Na occurs through the FS pathway (180-250 mumol/liter cell X h). The concentration of Ko at which the FS Na influx and efflux and the FS K influx and efflux become equal increased when Nai increased in high-K cells and when Ki was increased in high-Na cells. The net FS Na and K fluxes both approached zero at similar internal and external Na and K concentrations. In high-K cells, under conditions when net Na and K fluxes were near zero, the ratio of FS Na to FS K unidirectional flux was found to be 2:3. In high-K cells, the empirical expression (Nai/Nao)2(Ki/Ko)3 remained at constant value (apparent equilibrium constant, Kappeq +/- SEM = 22 +/- 2) for each set of internal and external cation concentrations at which there was no net Na flux. These results indicate that in the physiological region of concentrations of internal and external Na, K, and Cl, the stoichiometry of the FS Na and K fluxes is 2 Na:3 K. In high-Na cells under conditions when net FS Na and K fluxes were near zero, the ratio of FS Na to FS K unidirectional fluxes was 3:2 (1).(ABSTRACT TRUNCATED AT 400 WORDS)

We report in this paper different modes of Na and K transport in human red cells, which can be inhibited by furosemide in the presence of ouabain. Experimental evidence is provided for inward and outward coupled transport of Na and K, Ki/Ko and Nai/Nao exchange, and uncoupled Na or K efflux. The outward cotransport of Na and K was defined as the furosemide-sensitive (FS) component of Na and K effluxes into choline medium and as the Cl-dependent or cis-stimulated component of the ouabain-resistant (OR) Na and K effluxes. Inward cotransport of Na and K was defined by the stimulation by external Na (Nao) of the K influx and the stimulation by external K (Ko) of the Na influx in the presence of ouabain. Both effects were FS and Cl dependent. Experimental evidence for an FS Ki/Ko exchange pathway of the Na/K cotransport was provided by (a) the stimulation by external K of FS K influx and efflux, and (b) the stimulation by internal Na or K of FS K influx in the absence of external Na. Evidence for an FS Nai/Nao exchange pathway was provided by the stimulation of FS Na influx by internal Na from a K-free medium (130 mM NaCl). This pathway was four to six times smaller than the Ki/Ko exchange. In cells containing only Na or K, incubated in media containing only Na or K, respectively, there was FS efflux of the cation without simultaneous inward transport (FS uncoupled Na and K efflux). The stoichiometric ratio of FS outward cotransport of Na and K into choline medium varied with the ratio of Nai-to-Ki concentrations, and when Nai/Ki was close to 1, the ratio of FS outward Na to K flux was also 1. In choline media, FS Na efflux was inhibited by external K (noncompetitively), whereas FS k efflux was stimulated. The stimulation of FS K efflux was due to the stimulation by Ko of the Ki/Ko exchange pathway. Thus, the stoichiometry of FS Na and K effluxes also varied in the presence of external K. A minimal model for a reaction scheme of FS Na and K transport accounts for cis stimulation, trans inhibition, and trans stimulation, and for variable stoichiometry of the FS cation fluxes.

Cyclo(L-Lac-L-Val-D-Pro-D-Val)3 (PV-Lac) a structural analogue of the ion-carrier valinomycin, increases the cation permeability of lipid bilayer membranes by forming a 1:1 ion-carrier complex. The selectively sequence for PV-Lac is identical to that of valinomycin; i.e., Rb+ greater than K+ greater than Cs+ greater than or equal to NH+4 greater than Na+ greater than Li+. The steady-state zero-voltage conductance, G(0), is a saturating function of KCl concentration. A similar behavior was found for Rb+, Cs+, and NH+4. However, the ion concentration at which G(0) reaches a plateau strongly depends on membrane composition. The current-voltage curves present saturating characteristics, except at low ion concentrations of Rb+, K+, or Cs+. The ion concentration at which the saturating characteristics appear depends on membrane composition. These and other results presented in this paper agree with a model that assumes complexation between carrier and ion at the membrane-water interface. Current relaxation after voltage-jump studies were also performed for PV-Lac. Both the time constant and the amplitude of the current after a voltage jump strongly depend on ion concentration and membrane composition. These results, together with the stationary conductance data, were used to evaluate the rate constants of the PV-Lac-mediated K+ transport. In glycerolmonooleate they are: association rate constant, 2 x 10(6) M-1 s-1; dissociation rate constant, 4 x 10(5) s-1; translocation rate constant for complex, 5 x 10(4) s-1; and the rate of translocation of the free carrier (ks), 55 s-1. ks is much smaller for PV-Lac than for valinomycin and thus limits the efficiency with which the carrier is able to translocate cations across the membrane.

This paper reports the effects of peptide PV (primary structure: cyclo-(D-val-L-pro-L-val-D-pro)δ) on the electrical properties of sheep red cell lipid bilayers. The membrane conductance (Gm) induced by PV in either Na+ or K+ medium is proportional to the concentration of PV in the aqueous phase. The PV concentration required to produce a comparable increase in Gm in K+ medium is about 104 times greater than for its analogue, valinomycin (val). Although the selectivity sequence for PV and val is similar, K+ ≳ Rb+ > Cs+ > NH4+ > TI+ > Na+ > Li+; the ratio of GGm in K+ to that in Na+ is about 10 for PV compared to > 103 for val. When equal concentrations of PV are added to both sides of a bilayer, the membrane current approaches a maximum value independent of voltage when the membrane potential exceeds 100 mV. When PV is added to only one side of a bilayer separating identical salt solutions of either Na+ or K+ salts, rectification occurs such that the positive current flows more easily away rather than toward the side containing the carrier. Under these conditions, a large, stable, zero-current potential (VVm) is also observed, with the side containing PV being negative. The magnitude of this VVm is about 90 mV and relatively independent of PV concentration when the latter is larger than 2 Times; 10–5 M. From a model which assumes that Vm equals the equilibrium potential for the PV-cation complexes (MS+) and that the reaction between PV and cations is at equilibrium on the two membrane surfaces, we compute the permeability of the membrane to free PV to be about 10–5 cm s–1, which is about 10–7 times the permeability of similar membranes to free val. This interpretation is supported by the fact that the observed values of Vm are in agreement with the calculated equilibrium potential for MS+ over a wide range of ratios of concentrations of total PV in the two bathing solutions, if the unstirred layers are taken into account in computing the MS+ concentrations at the membrane surfaces.

Human cerebrospinal fluid (CSF) inhibits the Na+/K+ pump in human red cells and the activity of purified Na+/K+-ATPase (Halperin, J. A., Shaeffer, R., Galvez, L., and Malavé, S. (1985) Proc. Natl. Acad. Sci. U.S. A. 80, 6102-6104, 1983; Halperin, J. A., Martin, A. M., and Malavé, S. (1985) Life Sci. 37, 561-566. We describe here some properties of the CSF inhibitor of the Na+/K+ pump. Active material was extracted from human CSF with 50% methanol and then concentrated and desalted by ultrafiltration. This extract inhibited, in a dose-dependent manner, the ouabain-sensitive influx of K+ into human red cells and the activity of purified Na+/K+-ATPase. Partial separation of the inhibitory activity was achieved by gel filtration and reverse-phase high performance liquid chromatography. Inhibition of both pump and enzyme was specific in that other red cell membrane transport systems or enzymes examined were not influenced by CSF extracts. Dialysis and ultrafiltration experiments indicate that the molecular weight of the inhibitor is approximately equal to 600. The inhibitory activity is sensitive to proteolytic enzymes indicating that the inhibitor might be a small peptide. In the presence of CSF extract the K0.5 for external K+ to stimulate the Na+/K+ pump increased from 1.4 to 3.1 mM, suggesting that the CSF inhibitor competes with external K+ for stimulation of the pump. We estimate that the concentration of the inhibitor in CSF might be approximately equal to 50 pg/ml, a value close to the concentration of other active peptides found in human CSF.

We report here the effects of membrane potential on the permeability of the human erythrocyte to Na, K, and Ca. Membrane potential was changed either by varying the K concentration gradient in the presence of valinomycin or by varying the concentration gradient of the permeant anion nitrate in the presence of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. When the membrane potential was changed from inside negative (-10 mV) to inside positive (greater than 40 mV), influx, efflux, and net flux of Na and K increased. Marked net cation loss and cell shrinkage occurred in the absence of a chemical gradient for Na and K. This voltage-dependent increase in Na and K conductance is partially inhibited by 10 microM ruthenium red and persists when the membrane potential is returned to -10 mV after transient exposure to inside-positive potentials. A similar voltage-dependent behavior was found for Ca influx. The voltage-activated Ca influx is almost completely inhibited by 10 microM ruthenium red.

This paper describes the kinetics and stoichiometry of a tightly coupled Na-Li exchange transport system in human red cells. The system is inhibited by phloretin and furosemide but not by ouabain. Li influx by this system increases and saturates with increasing concentrations of external Li and internal Na and is inhibited competitively by external Na. Comparable functions relate Li efflux and Na efflux to internal and external Li and Na concentrations. Analysis of these relations yields the following values for the ion concentrations required to half-maximally activate the transport system: internal Na and Li 9.0 and 0.5 mM, respectively, external Na and Li 25 and 1.5 mM, respectively. The system performs a 1:1 exchange of Na and Li moving in opposite directions across the red cell membrane. We found no evidence for a simultaneous transport of more than one Na and Li by the system. The maximum transport rate of Na-dependent Li transport varied between 0.1 and 0.37 mmol/(liter of cells X h) in the red cells of the five normal male subjects studied. No significant variations between individual subjects were observed for bicarbonate-stimulated Li transport and for the residual Li fluxes which occur in the absence of bicarbonate and in the presence of ouabain plus phloretin.

In human red cells, Li is extruded against its own concentration gradient if the external medium contains Na as a dominant cation. This uphill net Li extrusion occurs in the presence of external Na but not K, Rb, Cs, choline, Mg, or Ca, is ouabain-insensitive, inhibited by phloretin, and does not require the presence of cellular ATP. Li influx into human red cells has a ouabain-sensitive and a ouabain-insensitive but phloretin-sensitive component. Ouabain-sensitive Li influx is competitively inhibited by external K and Na and probably involves the site on which the Na-K pump normally transports K into red cells. Ouabain does not inhibit Li efflux from red cells containing Li concentrations below 10 mM in the presence of high internal Na or K, whereas a ouabain-sensitive Li efflux can be measured in cells loaded to contain 140 mM Li in the presence of little or no internal Na or K. Ouabain-insensitive Li efflux is stimulated by external Na and not by K, Rb, Cs, choline, Mg, or Ca ions. Na-dependent Li efflux does not require the presence of cellular ATP and is inhibited by phloretin, furosemide, quinine, and quinidine. Experiments carried out in cells loaded in the presence of nystatin to contain either only K or only Na show that the ouabain-insensitive, phloretin-inhibited Li movements into or out of human red cells are stimulated by Na on the trans side and inhibited by Na on the cis side of the red cell membrane. The characteristics of the Na-dependent unidirectional Li fluxes and uphill Li extrusion are similar, suggesting that they are mediated by the same Na-Li countertransport system.