The influence of cytoplasmic sodium concentration on the stoichiometry of the sodium pump

Using inside-out vesicles of human red cell membranes, the effects of cytoplasmic Na+ in the range 0-5 mM on ATP-dependent 22Na+ influx (normal efflux) and 86Rb+ efflux (normal influx) were tested. The sodium pump stoichiometry, i.e. the ratio of net 22Na+ influx:86Rb+ efflux was reduced markedly when the cytoplasmic Na+ was reduced to less than 1 mM. Reduction in cytoplasmic Na+ concentration was associated also with a decreased sensitivity of the pump to effects of extracellular Rb+. Thus, extracellular (intravesicular) Rb+ stimulation observed at high ATP concentration and inhibition observed at low ATP concentration were not observed when the cytoplasmic (extravesicular) Na+ concentration was reduced to less than or equal to 0.2 mM. It is suggested that at low cytoplasmic Na+, the pump can operate with less than maximal sites filled with Na+ ions. Under this condition, it is likely that an enzymic step associated with either the ion translocation step or the enzyme's conformational transition becomes rate-limiting.     

Effects of cytoplasmic sodium concentration on the electrogenicity of the sodium pump

Inside-out membrane vesicles were prepared from human red blood cells pretreated with diisothiocyano-2,2'-disulfonic stilbene to inhibit anion fluxes. The pH-sensitive probe fluorescein isothiocyanate-dextran was incorporated inside the vesicles. Formation of pH gradients due to proton transport by the sodium pump was distinguished from pH gradients formed in response to transmembrane electrical potentials generated by the pump by virtue of their insensitivity and sensitivity, respectively, to dissipation by lipophilic cations. Under the conditions used (pH 6.6), proton transport by the Na,K-ATPase was minimized, and the formation of pH gradients in response to electrical potentials was detected. Thus, the generation of a strophanthidin-sensitive, ATP-dependent electrical potential, inside positive (approximately 1 mV) upon addition of 4 meq of sodium to potassium-filled inside-out vesicles is consistent with the well documented stoichiometry of three sodium ions exchanging with two potassium ions. In contrast, when the cytoplasmic sodium concentration is reduced to less than or equal to 0.4 mM, the potential generated is of the opposite sign, i.e. inside negative, consistent with the decreased Na:K coupling ratio reported previously, i.e. Na:K(Rb) coupling ratios of approximating 1:2 when the sodium concentration is reduced to 0.2 mM (Blostein, R. (1983) J. Biol. Chem. 258, 12228-12232).     

Sodium pump-catalyzed sodium-sodium exchange associated with ATP hydrolysis

Inside-out red cell membrane vesicles have been used to study sodium interactions with the ATP-dependent sodium pump at sites accessible to both membrane surfaces. ATP-dependent 22Na+ influx (equivalent to efflux from cells) shows sigmoid dependence on extravesicular Na+ concentration. This is observed both in the absence of intravesicular cations and in the presence of intravesicular K or Rb ions. The kinetic behavior is similar to that observed earlier with intact cells, (Garay, R. P., and Garrahan, P. J. (1973) J. Physiol. (Lond.) 231, 297-325) and is consistent with a ratio of close to three Na ions transported per molecule of ATP hydrolyzed. With vesicles having relatively high intravesicular sodium concentration, (approximately 50 mM NaCl), the sodium pump effects an ATP-dependent sodium efflux coupled to sodium influx and to strophanthidin-sensitive ATP hydrolysis. The influx:efflux stoichiometry is approximately 1:1, and the influx:ATP hydrolysis ratio is close to 3. This ATP-dependent exchange has a higher affinity for vanadate than ATP plus ADP-dependent sodium exchange. It is concluded that this sodium-sodium exchange mode resembles sodium-potassium exchange whereby intravesicular sodium, i.e. sodium at the extracellular surface, at relatively high concentration, behaves like potassium.     

Transmembrane effects in the sodium pump system. I. The effect of external potassium and rubidium on the dependence of sodium efflux on sodium concentration in the frog muscle

The dependence of sodium efflux on intracellular sodium content with various potassium and rubidium concentration in the external medium has been studied on frog sartorious muscle. In potassium-sodium-free magnesium medium ouabain-sensitive sodium efflux was shown to be proportional to internal sodium concentration. In the presence of external ribidium (0.5--5.0 mM) the efflux concentration relations are non-linear, being closely described by assuming that 3 Na+ are transported per pump cycle. In sodium loaded muscles the efflux concentration curve was found to be dependent on the external rubidium concentration, becoming linear instead of S-shaped with the decrease in internal rubidium concentration from 5.0--2.5 to 1.0--0.5 mM. The apparent affinity constant for the internal sodium pump site increased with increasing the external rubidium (potassium) concentration. The data obtained may contribute to the kinetic evaluation of the type of Na-K pump mechanism, being more consistent with simultaneous model of pump operation.     

Stoichiometry of sodium-calcium exchange in cardiac sarcolemmal vesicles. Coupling to the sodium pump.

Vesicles isolated from cardiac muscle exhibited Na,Ca exchange activity which can be measured by 45Ca influx or efflux of by 22Na efflux. The stoichiometry of Na,Ca exchange was 3 Na:1 Ca. These vesicles also exhibited ATP-dependent 22Na transport which was inhibited by ouabain indicating that this activity is due to the sodium pump, an activity which is thought to reside only in the sarcolemma. The addition of calcium caused rapid efflux of 22Na from vesicles loaded by ATP-dependent 22Na uptake indicating that the Na,Ca exchange is located in the same vesicles as the sodium pump and is thus also a sarcolemmal activity.     

Generation of a membrane potential by sodium-dependent succinate efflux in Selenomonas ruminantium.

When Selenomonas ruminantium HD4 was grown in a chemostat, maximal succinate production and the highest molar growth yield values were both observed at a dilution rate of roughly 0.2 h-1. To determine the possible relationship between succinate efflux and high molar growth yields, the generation of a membrane potential by succinate efflux was studied in whole cells and vesicles (inside-out and right-side-out) prepared from S. ruminantium. Washed whole cells took up succinate in the absence of an exogenous energy supply; uptake was completely abolished by brief treatment with dinitrophenol or with nigericin and valinomycin. High levels of sodium ions (with respect to the intracellular sodium concentration in the assay buffer had a stimulatory effect on succinate uptake. When succinate was added to inside-out vesicles, a membrane potential (inside positive) was generated, as indicated by fluorescence quenching of the anionic lipophilic dye Oxonol V. Fluorescence quenching was sensitive to uncoupling by gramicidin D but only partially sensitive to the uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. In right-side-out vesicles, succinate uptake could be driven by an artificially imposed sodium gradient but not by a potassium diffusion potential; imposition of both a sodium gradient and potassium diffusion potential resulted in improved succinate uptake. The generation of a membrane potential (inside negative) upon succinate efflux was demonstrated directly in right-side-out vesicles when succinate-loaded vesicles were diluted into succinate-free buffer, and the lipophilic cationic probe tetraphenylphosphonium accumulated in the vesicles. Results indicate that an electrogenic succinate-sodium symporter is present in S. ruminantium. Transport of succinate out of the cell via the symporter might be responsible for the high molar growth yields obtained by this organism when it is grown at dilution rates where maximal succinate production occurs.     

Formation of inside-out vesicles of Bacillus licheniformis. Dependence on buffer composition and lysis procedure.

1. The extent to which the cytoplasmic membrane of the Gram-positive bacterium Bacillus licheniformis formed inside-out vesicles was studied with the freeze-fracture technique. The membrane orientation appeared to be dependent on the buffer compositon as well as on the lysis procedure used. 2. By manipulating these conditions, membrane preparations were obtained with the percentage of inside-out vesicles varying from 15 to 80%. 3. More vesicles had the opposite orientation when the cells were lysed in potassium phosphate buffer than when they were lysed in sodium phosphate buffer. Tris-HCl buffer favoured the formation of inside-out vesicles more than phosphate buffer. 4. Lysis of protoplasts in hypotonic buffers resulted in more inside-out vesicles than did direct lysis of cells in hypotonic media. 5. In an attempt to explain the observed differences, experiments were performed in which the morphology of thin-sectioned lysing cells in sodium phosphate buffer was compared with that in potassium phosphate buffer. The results from these experiments indicate that the formation of inside-out vesicles is brought about by an effect on the membrane itself rather than on the cell wall, on the cell wall membrane association, or on the cytoplasm.     

Interaction of asparagine-linked oligosaccharides with an immobilized rice (Oryza sativa) lectin column.

Inside-out plasma-membrane vesicles isolated from rat liver [Prpic, Green, Blackmore & Exton (1984) J. Biol. Chem. 259, 1382-1385] accumulated a substantial amount of 45Ca2+ when they were incubated in a medium whose ionic composition and pH mimicked those of cytosol and which contained MgATP. The Vmax of the initial 45Ca2+ uptake rate was 2.9 +/- 0.6 nmol/min per mg and the Km for Ca2+ was 0.50 +/- 0.08 microM. The ATP-dependent 45Ca2+ uptake by inside-out plasma-membrane vesicles was about 20 times more sensitive to saponin than was the ATP-dependent uptake by a microsomal preparation. The 45Ca2+ efflux from the inside-out vesicles, which is equivalent to the Ca2+ influx in intact cells, was increased when the free Ca2+ concentration in the medium was decreased. The Ca2+ antagonists La3+ and Co2+ inhibited the 45Ca2+ efflux from the vesicles. Neomycin stimulated the Ca2+ efflux in the presence of either a high or a low free Ca2+ concentration. These results confirm that polyvalent cations regulate Ca2+ fluxes through the plasma membrane.     

Energy-dependent export of the 13-oxooctadecadienoic acid-glutathione conjugate from HT-29 cells and plasma membrane vesicles

Numerous studies have identified members of the multidrug resistance protein (MRP) family of ABC transporters as ATP-dependent GS-X pumps responsible for export of various xenobiotic conjugates, and the few known glutathione conjugates of endogenous metabolites. In the present study we have investigated the possibility that the glutathione conjugate of 13-oxooctadecadienoic acid (13-OXO-SG), is exported from HT-29 cells by one of these GS-X pumps. The precursor 13-oxooctadecadienoic acid (13-OXO) is a metabolic oxidation product of linoleic acid. The transport of 13-OXO-SG is compared to that of the glutathione conjugate of chlorodinitrobenzene (DNP-SG). The results show that the efflux of 13-OXO-SG is ATP-dependent. In cultured HT-29 cells as well as in inside-out vesicles prepared from these cells, significant inhibition of conjugate export is achieved by the energy disrupters, beta,gamma-methylene ATP, sodium vanadate, and 2-deoxyglucose. Significant inhibition of the vesicle-mediated transport is also observed in the presence of genistein and verapamil. In inside-out vesicles, the transport of both conjugates exhibits saturation with an apparent K(m) of 325.5 microM and a V(max) of 0.0669 nmol/mg protein per min for 13-OXO-SG and a K(m) of 169 microM and a V(max) of 0.496 nmol/mg protein per min for DNP-SG. Furthermore, co-inhibition is observed when both conjugates are present simultaneously which is consistent with the involvement of common pumps. The data in this report demonstrate the involvement of an ATP-dependent pump in the metabolic disposition of endogenously derived metabolites of linoleic acid.     

Sodium-calcium exchange in transverse tubules isolated from frog skeletal muscle

Transverse tubule vesicles isolated from frog skeletal muscle display sodium-calcium exchange activity, which was characterized measuring 45Ca influx in vesicles incubated with sodium. The initial rates of exchange varied as a function of the membrane diffusion potentials imposed across the membrane vesicles, increasing with positive intravesicular potentials according to an electrogenic exchange with a stoichiometry greater than 2 sodium ions per calcium ion transported. The exchange activity was a saturable function of extravesicular free calcium, with an apparent K0.5 value of 3 microM and maximal rates of exchange ranging from 3 to 5 nmol/mg protein per 5 s. The exchange rate increased when intravesicular sodium concentration was increased; saturation was approached when vesicles were incubated with concentrations of 160 mM sodium. The isolated transverse tubule vesicles, which are sealed with the cytoplasmic side out, had a luminal content of 112 +/- 39 nmol calcium per mg protein. In the absence of sodium, the exchanger carried out electroneutral calcium-calcium exchange, which was stimulated by increasing potassium concentrations in the intravesicular side. Calcium-calcium exchange showed an extravesicular calcium dependence similar to the calcium dependence of the sodium-calcium exchange, with an apparent K0.5 of 6 microM. Sodium-calcium and calcium-calcium exchange were both inhibited by amiloride. The sodium-calcium exchange system operated both in the forward and in the reverse mode; sodium, as well as calcium, induced calcium efflux from 45Ca-loaded vesicles. This system may play an important role in decreasing the intracellular calcium concentration in skeletal muscle following electrical stimulation.     

Symmetry properties of the Na+-Ca2+ exchange mechanism in cardiac sarcolemmal vesicles

The Na+-Ca2+ exchange mechanism in cardiac sarcolemmal vesicles can catalyze the exchange of Ca2+ on either side of the sarcolemmal membrane for Na+ on the opposing side. Little is known regarding the relative affinities of Na+ and Ca2+ for exchanger binding sites on the intra- and extracellular membrane surfaces. We have previously reported (Philipson, K.D. and Nishimoto, A.Y. (1982) J. Biol. Chem. 257, 5111-5117) a method for measuring the Na+-Ca2+ exchange of only the inside-out vesicles in a mixed population of sarcolemmal vesicles (predominantly right-side-out). We concluded that the apparent Km(Ca2+) for Na+i-dependent Ca2+ uptake was similar for inside-out and right-side-out vesicles. In the present study, we examine in detail Na+o-dependent Ca2+ efflux from both the inside-out and the total population of vesicles. To load vesicles with Ca2+ prior to measurement of Ca2+ efflux, four methods are used: 1, Na+-Ca2+ exchange; 2, passive Ca2+ diffusion; 3, ATP-dependent Ca2+ uptake; 4, exchange of Ca2+ for Na+ which has been actively transported into vesicles by the Na+ pump. The first two methods load all sarcolemmal vesicles with Ca2+, while the latter two methods selectively load inside-out vesicles with Ca2+. We are able to conclude that the dependence of Ca2+ efflux on the external Na+ concentration is similar in inside-out and right-side-out vesicles. Thus the apparent Km(Na+) values (approximately equal to 30 mM) of the Na+-Ca2+ exchanger are similar on the two surfaces of the sarcolemmal membrane. In other experiments, external Na+ inhibited the Na+i-dependent Ca2+ uptake of the total population of vesicles much more potently than that of the inside-out vesicles. Apparently Na+ can compete for the Ca2+ binding site more effectively on the external surface of right-side-out than on the external surface of inside-out vesicles. Thus, although affinities for Na+ or Ca2+ (in the absence of the other ion) appear symmetrical, the interactions between Na+ and Ca2+ at the two sides of the exchanger are not the same. The Na+-Ca2+ exchanger is not a completely symmetrical transport protein.     

Energy-dependent efflux of methotrexate in L1210 leukemia cells. Evidence for the role of an ATPase obtained with inside-out plasma membrane vesicles.

Earlier studies from our laboratory (Dembo, M., Sirotnak F. M., and Moccio, D. M. (1984) J. Membr. Biol. 78, 9-17) suggested that methotrexate (MTX) efflux from L1210 cells was mediated predominantly by an ATP-dependent, outwardly directed, mechanism. To examine this process further, we utilized predominantly (74%) inside-out plasma membrane vesicle preparations derived from an L1210 cell variant (L1210/R24) with 15-fold reduced Vmax for [3H]MTX influx. Efflux of [3H]MTX, under nonionic buffer conditions, in these inside-out membrane vesicles was temperature and ATP dependent (apparent Km = 0.40 +/- 0.06 mM), osmotically sensitive, and unaffected by protonophores. The presence of K+, Na+, Cl-, and HCO3- at their physiological concentrations had no effect on [3H]MTX efflux. Other triphosphonucleotides (GTP and CTP), but not a nonhydrolyzable analogue, adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S), could also stimulate efflux, but to a lesser extent. Also, ATP gamma S and orthovanadate were potent inhibitors of ATP-dependent efflux of [3H]MTX. Other experiments revealed a system with low saturability for [3H]MTX during efflux (apparent Km = 46 +/- 7 microM), but extremely high capacity (106 +/- 15 pmol/min/mg protein), and a pH optimum in the range of 5.5-6. However, appreciable efflux was measured in the physiological range of pH 6.7-6.9. A number of inhibitors or copermeants for ATP-dependent [3H]MTX efflux in intact L1210 cells were inhibitors of ATP-dependent efflux in inside-out plasma membrane vesicles, including, cholate, bromosulfophthalein, verapamil, quinidine, and reserpine. These findings and other results showing that bromosulfophthalein will completely inhibit efflux are consistent with a role for an ATPase in [3H]MTX efflux, and suggest that the process under study is the bromosulfophthalein-sensitive, ATP-dependent route responsible for the majority of [3H]MTX efflux in intact L1210 cells.     

Transmembrane effects in the sodium pump system. II. The ratio of the sodium efflux to the sodium concentration in frog muscle in media with various sodium substitutes

The dependence of sodium efflux on the internal sodium concentration on sodium-free magnesium, Tris, coline and lithium media was investigated on frog striated muscle. In all the sodium-substituted media, the efflux concentration curve was found to be dependent on the external rubidium concentration, being S-shaped at the saturating external rubidium (potassium) concentration and becoming close to linear at the low external rubidium concentration (0.5-1.0 microM). The maximal sodium efflux at saturating levels of internal sodium concentrations remains unchanged with various sodium substitutes in the medium, whereas the affinity constant of internal sodium sites is dependent on the external cations.     

Beta-alanine transport in synaptic plasma membrane vesicles from rat brain. Efflux, exchange and stoichiometry

The efflux and exchange of beta-alanine were studied in synaptic plasma membrane vesicles from rat brain. The mechanism of beta-alanine translocation has been probed by comparing the ion dependence of net efflux to that of exchange. Dilution-induced efflux requires the simultaneous presence of internal sodium and chloride ions while influx is dependent on the presence of these two ions on the outside [Zafra, F., Aragón, M. C., Valdivieso, F. and Giménez, C. (1984) Neurochem Res. 9, 695-707]. These data show that the release of beta-alanine occurs via the carrier system and that it is cotransported with sodium and chloride ions. beta-Alanine efflux from the membrane vesicles is stimulated by external beta-alanine. This exchange does not require external sodium and chloride but it is dependent on the external concentration of beta-alanine. Half-maximal stimulation is obtained at a beta-alanine concentration similar to the Km for beta-alanine influx. Results of the direct measurements of the coupling of sodium and chloride to the transport of beta-alanine by using a kinetic approach allow us to propose a stoichiometry for the translocation cycle catalyzed by the beta-alanine transporter of three sodium ions and one chloride ion per beta-alanine zwitterion. To account for all the observed effects of external ions, beta-alanine concentrations and membrane potential on beta-alanine influx and efflux, a kinetic model of the Na+/Cl-/beta-alanine cotransport system is discussed.     

The kinetics of sodium extrusion in striated muscle as functions of the external sodium and potassium ion concentrations

After a 20 min initial washout, the rate of loss of radioactively labeled sodium ions from sodium-enriched muscle cells is sensitive to the external sodium and potassium ion concentrations. In the absence of external potassium ions, the presence of external sodium ions increases the sodium efflux. In the presence of external potassium ions, the presence of external sodium ions decreases the sodium efflux. In the absence of external potassium ions about one-third of the Na⁺ efflux that depends upon the external sodium ion concentration can be abolished by 10^-5 M glycoside. The glycoside-insensitive but external sodium-dependent Na⁺ efflux is uninfluenced by external potassium ions. In the absence of both external sodium and potassium ions the sodium efflux is relatively insensitive to the presence of 10^-5 M glycoside. The maximal external sodium-dependent sodium efflux in the absence of external potassium ions is about 20% of the magnitude of the maximal potassium-dependent sodium efflux. The magnitude of the glycoside-sensitive sodium efflux in K-free Ringer solution is less than 10% of that observed when sodium efflux is maximally activated by potassium ions. The inhibition of the potassium-activated sodium efflux by external sodium ions is of the competitive type. Reducing the external sodium ion concentration displaces the plots of sodium extrusion rate vs. [K]_o to the left and upwards.     

Energy-dependent export of the 13-oxooctadecadienoic acid–glutathione conjugate from HT-29 cells and plasma membrane vesicles

Numerous studies have identified members of the multidrug resistance protein (MRP) family of ABC transporters as ATP-dependent GS-X pumps responsible for export of various xenobiotic conjugates, and the few known glutathione conjugates of endogenous metabolites. In the present study we have investigated the possibility that the glutathione conjugate of 13-oxooctadecadienoic acid (13-OXO-SG), is exported from HT-29 cells by one of these GS-X pumps. The precursor 13-oxooctadecadienoic acid (13-OXO) is a metabolic oxidation product of linoleic acid. The transport of 13-OXO-SG is compared to that of the glutathione conjugate of chlorodinitrobenzene (DNP-SG). The results show that the efflux of 13-OXO-SG is ATP-dependent. In cultured HT-29 cells as well as in inside-out vesicles prepared from these cells, significant inhibition of conjugate export is achieved by the energy disrupters, β,γ-methylene ATP, sodium vanadate, and 2-deoxyglucose. Significant inhibition of the vesicle-mediated transport is also observed in the presence of genistein and verapamil. In inside-out vesicles, the transport of both conjugates exhibits saturation with an apparent K_m of 325.5 μM and a V_max of 0.0669 nmol/mg protein per min for 13-OXO-SG and a K_m of 169 μM and a V_max of 0.496 nmol/mg protein per min for DNP-SG. Furthermore, co-inhibition is observed when both conjugates are present simultaneously which is consistent with the involvement of common pumps. The data in this report demonstrate the involvement of an ATP-dependent pump in the metabolic disposition of endogenously derived metabolites of linoleic acid.     

The stoichiometry of the Ca2+ pump in human erythrocyte vesicles: modulation by Ca2+, Mg2+ and calmodulin

Active Ca2+ uptake and the associated (Ca2+ + Mg2+)-ATPase activity were studied under the same conditions in an inside-out vesicle preparation of human red blood cells made essentially by the procedure of Quist and Roufogalis (Journal of Supramolecular Structure 6, 375-381, 1977). Some preparations were treated with 1 mM EDTA at 30 degrees to further deplete them of endogenous levels of calmodulin. As the Ca2+ taken up by the EDTA-treated inside-out vesicles, as well as the non-EDTA treated vesicles, was maintained after addition of 4.1 mM EGTA, the vesicles were shown to be impermeable to the passive leak of Ca2+ over the time course of the experiments. In the absence of added calmodulin, both active Ca2+ uptake and (Ca2+ + Mg2+)-ATPase were sensitive to free Ca2+ over a four log unit concentration range (0.7 microM to 300 microM Ca2+) at 6.4 mM MgCl2. Below 24 microM Ca2+ the stoichiometry of calcium transported per phosphate liberated was close to 2:1, both in EDTA and non-EDTA treated vesicles. Above 50 microM Ca2+ the stoichiometry approached 1:1. When MgCl2 was reduced from 6.4 mM to 1.0 mM, the stoichiometry remained close to 2:1 over the whole range of Ca2+ concentrations examined. In contrast to the results at 6.4 mM MgCl2, the Ca2+ pump was maximally activated at about 2 microM free Ca2+ and significantly inhibited above this concentration at 1 mM MgCl2. Calmodulin (0.5-2.0 microgram/ml) had little effect on the stoichiometry in any of the conditions examined. The possible significance of a variable stoichiometry of the Ca2+ pump in the red blood cell is discussed.     

The function of Gp170, the multidrug resistance gene product, in rat liver canalicular membrane vesicles

Gp170 (also known as P-glycoprotein) is a transmembrane glycoprotein which is overexpressed in multidrug-resistant tumor cells and is also found in the apical plasma membrane domain of several normal human and animal tissues. Gp170 has been postulated to function as an energy-dependent efflux pump for cytotoxic drugs. In rat liver, Gp170 is restricted to the bile canalicular domain of the plasma membrane. Canalicular membrane vesicles (CMV), but not sinusoidal membrane vesicles, contained a approximately 160-kDa protein which reacts with anti-Gp170 monoclonal antibody and manifest ATP-dependent [3H]daunomycin transport which is temperature dependent, osmotically sensitive, and saturable. Among several nucleotides, ATP was a potent stimulator of transport whereas non- or slowly hydrolyzable analogues (adenosin-5-O-(3-thiotriphosphate, adenyl-5-yl-imidodiphosphate) were ineffective. ATP-dependent daunomycin transport was inhibited by cytotoxic drugs (vinblastine, vincristine, and adriamycin) and other drugs, such as verapamil and quinidine, which restore anti-cancer drug sensitivity in resistant cells. Inside-out CMV were separated from right side-out CMV by antibody-induced affinity density perturbation. Only inside-out CMV manifested ATP-dependent daunomycin transport. These results suggest that Gp170 is an ATP-dependent efflux pump which is responsible for the undirectional, energy-dependent transport of daunomycin and other drugs by rat liver into the bile.     

Effect of gramicidin A and thallium ions on cation effluxes in frog sartorius muscle

The effluxes of potassium, rubidium, sodium and lithium from the sartorius muscle of Rana temporaria in magnesium-Ringer solution free of sodium and potassium have been studied with the flame-emission technique. The channel-forming antibiotic gramicidin A (2.5 X X10(-7)-1 X 10(-6) mol/l) enhanced the efflux of potassium and rubidium and increased the rate constants of these effluxes. Gramicidin had small if any effect on sodium and lithium effluxes and rate constants. After 60-100 min in a gramicidin-containing medium, the potassium efflux and the corresponding rate constant reached a steady-state level. This steady-state value depended on gramicidin concentration. Effect of gramicidin on both the potassium efflux and the rate constant was partially reversible. Thallium ions (2.5 X 10(-3) and 5 X 10(-3) mol/l) in sodium- and potassium- free magnesium Ringer solution caused a large increase in effluxes of all the cations examined (K+, Rb+ and Na+) both in presence and absence of gramicidin. Possible mechanisms of gramicidin and thallium effects are discussed.