Unconventional Chemiosmotic Coupling of NHA2, a Mammalian Na+/H+ Antiporter,to a Plasma Membrane H+ Gradient

Human NHA2, a newly discovered cation proton antiporter, is implicated in essential hypertension by gene linkage analysis. We show that NHA2 mediates phloretin-sensitive Na⁺-Li⁺ counter-transport (SLC) activity, an established marker for hypertension. In contrast to bacteria and fungi where H⁺ gradients drive uptake of metabolites, secondary transport at the plasma membrane of mammalian cells is coupled to the Na⁺ electrochemical gradient. Our findings challenge this paradigm by showing coupling of NHA2 and V-type H⁺-ATPase at the plasma membrane of kidney-derived MDCK cells, resulting in a virtual Na⁺ efflux pump. Thus, NHA2 functionally recapitulates an ancient shared evolutionary origin with bacterial NhaA. Although plasma membrane H⁺ gradients have been observed in some specialized mammalian cells, the ubiquitous tissue distribution of NHA2 suggests that H⁺-coupled transport is more widespread. The coexistence of Na⁺ and H⁺-driven chemiosmotic circuits has implications for salt and pH regulation in the kidney.     

Role of Na+/Ca2+ exchange and the plasma membrane Ca2+ pump in hormone-mediated Ca2+ efflux from pancreatic acini

Summary The relative contributions of the Na⁺/Ca²⁺ exchange and the plasma membrane Ca²⁺ pump to active Ca²⁺ efflux from stimulated rat pancreatic acini were studied. Na⁺ gradients across the plasma membrane were manipulated by loading the cells with Na⁺ or suspending the cells in Na⁺-free media. The rates of Ca²⁺ efflux were estimated from measurements of [Ca²⁺] _i using the Ca²⁺-sensitive fluorescent dye Fura 2 and⁴⁵Ca efflux. During the first 3 min of cell stimulation, the pattern of Ca²⁺ efflux is described by a single exponential function under control, Na⁺-loaded, and Na⁺-depleted conditions. Manipulation of Na⁺ gradients had no effect on the hormone-induced increase in [Ca²⁺] _i . The results indicate that Ca²⁺ efflux from stimulated pancreatic acinar cells is mediated by the plasma membrane Ca²⁺ pump. The effects of several cations, which were used to substitute for Na⁺, on cellular activity were also studied. Choline⁺ and tetramethylammonium⁺ (TMA⁺) released Ca²⁺ from intracellular stores of pancreatic acinar, gastric parietal and peptic cells. These cations also stimulated enzyme and acid secretion from the cells. All effects of these cations were blocked by atropine. Measurements of cholecystokinin-octapeptide (CCK-OP)-stimulated amylase release from pancreatic acini, suspended in Na⁺, TMA⁺, choline⁺, or N-methyl-d-glucamine⁺ (NMG⁺) media containing atropine, were used to evaluate the effect of the cations on cellular function. NMG⁺, choline⁺, and TMA⁺ inhibited amylase release by 55, 40 and 14%, respectively. NMG⁺ also increased the Ca²⁺ permeability of the plasma membrane. Thus, to study Na⁺ dependency of cellular function, TMA⁺ is the preferred cation to substitute for Na⁺. The stimulatory effect of TMA⁺ can be blocked by atropine.     

Sodium-calcium ion exchange in skeletal muscle sarcolemmal vesicles

Summary The Ca²⁺ permeability of rabbit skeletal muscle sarcolemmal vesicles was investigated by means of radioisotope flux measurements. A membrane vesicle fraction highly enriched in sarcolemma, as revealed by enzymatic markers, was obtained from the 22–27% region of sucrose gradients after isopycnic centrifugation. The ability of sarcolemmal vesicles to exchange Na⁺ for Ca²⁺ was investigated by measuring Ca²⁺ influx into and efflux from sarcolemmal vesicles in the presence and absence of a Na⁺ gradient. It was found that Ca²⁺ movements were enhanced in the direction of the higher Na⁺ concentration. When intra- and extravesicular Na⁺ concentrations were high, Na⁺–Na⁺ exchange predominated and Na⁺–Ca²⁺ exchange was low or absent. The presence of the Ca²⁺ ionophore A23187 in the dilution medium resulted in the rapid release of Ca²⁺ and the elimination of the Na⁺-enhanced efflux of Ca²⁺, suggesting that internal rather than bound external Ca²⁺ was exchanged with Na⁺. La³⁺ abolished Na⁺–Ca²⁺ exchange and decreased overall membrane permeability. Na⁺–Ca²⁺ exchange was not due to sarcoplasmic reticulum or mitochondrial contaminants. This investigation suggests that skeletal muscle, like cardiac muscle and neurons, is capable of a transmembranous Na⁺–Ca²⁺ exchange.     

Transport in halobacterium halobium: Light-induced cation-gradients,amino acid transport kinetics,and properties of transport carriers

Cell envelope vesicles prepared from H. halobium contain bacteriorhodopsin and upon illumination protons are ejected. Coupled to the proton motive force is the efflux of Na⁺. Measurements of ²²Na flux, exterior pH change, and membrane potential, ΔΨ (with the dye 3,3′-dipentyloxadicarbocyanine) indicate that the means of Na⁺ transport is sodium/proton exchange. The kinetics of the pH changes and other evidence suggests that the antiport is electrogenic (H⁺/Na⁺ > 1). The resulting large chemical gradient for Na⁺ (outside > inside), as well as the membrane potential, will drive the transport of 18 amino acids. The 19th, glutamate, is unique in that its accumulation is indifferent to ΔΨ: this amino acid is transported only when a chemical gradient for Na⁺ is present. Thus, when more and more NaCl is included in the vesicles glutamate transport proceeds with longer and longer lags. After illumination the gradient of H⁺ collapses within 1 min, while the large Na⁺ gradient and glutamate transporting activity persists for 10–15 min, indicating that proton motive force is not necessary for transport. A chemical gradient of Na⁺, arranged by suspending vesicles loaded with KCl in NaCl, drives glutamate transport in the dark without other sources of energy, with V_max and K_m comparable to light-induced transport. These and other lines of evidence suggest that the transport of glutamate is facilitated by symport with Na⁺, in an electrically neutral fashion, so that only the chemical component of the Na⁺ gradient is a driving force. The transport of all amino acids but glutamate is bidirectional. Actively driven efflux can be obtained with reversed Na⁺ gradients (inside > outside), and passive efflux is considerably enhanced by intravesicle Na⁺. These results suggest that the transport carriers are functionally symmetrical. On the other hand, noncompetitive inhibition of transport by cysteine (a specific inhibitor of several of the carriers) is only obtained from the vesicle exterior and only for influx: these results suggest that in some respects the carriers are asymmetrical. A protein fraction which binds glutamate has been found in cholate-solubilized H. halobium membranes, with an apparent molecular weight of 50,000. When this fraction (but not the others eluted from an Agarose column) is reconstituted with soybean lipids to yield lipoprotein vesicles, facilitated transport activity is regained. Neither binding nor reconstituted transport depend on the presence of Na⁺. The kinetics of the transport and of the competitive inhibition by glutamate analogs suggest that the protein fraction responsible is derived from the intact transport system.     

The Na+ gradient hypothesis in cytoplasts derived from Ehrlich ascites tumor cells

The concentration gradients of Na⁺ and the non-metabolizable amino acid, α-aminoisobutyric acid, and the membrane potential were measured in cytoplasts derived from Ehrlich ascites tumor cells in order to test the Na⁺ gradient hypothesis for the active transport of neutral amino acids in animal cells. According to this hypothesis, the Na⁺ electrochemical gradient and the amino acid activity gradient should be equal at the steady state. It has been difficult to measure the Na⁺ electrochemical gradient in intact Ehrlich cells because Na⁺ may be sequestered in the nuclei of these cells. This problem is avoided with cytoplasts derived from Ehrlich cells because they do not contain internal compartments where Na⁺ could be sequestered. Since these cytoplasts also maintain steady state concentrations of Na⁺, K⁺, and α-aminoisobutyric acid similar to those found in whole Ehrlich cells, they are uniquely suited for testing the Na⁺ gradient hypothesis. Assuming the activity coefficients of external and cytoplasmic Na⁺ are equal, the energy in the Na⁺ electrochemical gradient of cytoplasts was 90% of that in the α-aminoisobutyric acid concentration gradient at the steady state. If the Na⁺ gradient hypothesis is correct, the 10% difference between these two gradients cannot be explained in terms of the sequestration of Na⁺ in the nucleus because cytoplasts do not contain internal compartments.     

Activation of Na+,H+-Exchange in Erythrocytes of the River Lamprey Lampetra fluviatilis

To study H⁺ transport, the lamprey red blood cells were acidified to pH 6.0 by a pretreatment with an ionophore, nigericin. Incubation of the acidified cells in NaCl-medium at pH 8.0 was accompanied by a rapid H⁺ efflux from the erythrocytes. There was a tenfold decrease of the H⁺ efflux rate on addition to NaCl-medium of dimethylamiloride or on replacing Na⁺ in the medium (KCl-medium, pH 8.0). A high rate of Na+ influx into the acidified erythrocytes occurred only in the presence of H⁺ gradient (pH medium 8.0), but not in its absence (pH medium 6.0). The Na⁺-dependent H⁺ efflux from the cells and H⁺-dependent Na⁺ influx into the cells were quantitatively similar (about 700 mmol/l cells/h). A rapid elevation of the intracellular Na⁺ concentration as measured by flame photometry was also observed during incubation of the acidified cells in NaCl-medium (pH 8.0). The H⁺-dependent Na⁺ influx and an increase of the Na⁺ content in the acidified cells were significantly inhibited by amiloride. The data obtained for the first time prove with certainty the presence of the Na⁺/H⁺ exchanger in erythrocytes of the river lamprey.     

Functional characterization of the uncoupler-insensitive Na+ pump of the halotolerant bacterium,Ba1

Respiration initiates Na⁺ efflux from Na⁺-preloaded cells of the halotolerant bacterium, Ba₁. This efflux can take place against the concentration and electrochemical gradients. Since it is not inhibited by carbonylcyanide-p-trifluoromethoxyphenyl-hydrazone or N'N'-dicyclohexylcarbodiimide, it seems unlikely that either Δp (electrochemical potential difference of H⁺ across the membrane) generated by the primary proton pump or ATP play a role in the transduction of the energy supplied by electron transport. The electrogenic extrusion of Na⁺ causes passive counterflow of protons and/or simultaneous flux of permeant anions. In the absence of permeant anions the charge compensation attained by influx of protons is not complete. The membrane potential which persists in this case is inside negative and insensitive to uncoupler. The influx of protons builds up a ΔpH of reversed sign (more acid inside), which is insensitive to uncoupler. The simultaneous efflux of Na⁺ and permeant anions diminishes the intracellular salt content and, as a corollary, causes volume contraction. Thus, the respiration-linked, uncoupler-insensitive Na⁺ pump may play a role in the regulation of the intracellular salt content.     

In Vivo pH Regulation by a Na/H Antiporter in the Halotolerant Alga Dunaliella salina

Na⁺/H⁺ exchange activity in whole cells of the halotolerant alga Dunaliella salina can be elicited by intracellular acidification due to addition of weak acids at appropriate external pH. The changes in both intracellular pH and Na⁺ were followed. Following a mild intracellular acidification, intracellular Na⁺ content increased dramatically and then decreased. We interpret the phase of Na⁺ influx as due to the activation of the plasma membrane Na⁺/H⁺ antiporter and the phase of Na⁺ efflux as due to an active Na⁺ extrusion process. The following observations are in agreement with this interpretation: (a) the Na⁺ influx phase was sensitive to Li⁺, which is an inhibitor of the Na⁺/H⁺ antiporter, did not require energy, and was insensitive to vanadate; (b) the Na⁺ efflux phase is energy-dependent and sensitive to the plasma membrane ATPase inhibitor, vanadate. Following intracellular acidification, a drastic decrease in the intracellular ATP content is observed that is reversed when the cells regain their neutral pH value. We suggest that the intracellular acidification-induced change in the internal Na⁺ concentration is due to a combination of Na⁺ uptake via the Na⁺/H⁺ antiporter and an active, ATPase-dependent, Na⁺ extrusion. The Na⁺/H⁺ antiporter seems, therefore, to play a principal role in internal pH regulation in Dunaliella.     

Sodium-dependent Potassium Channels in Leech P Neurons

In leech P neurons the inhibition of the Na⁺-K⁺ pump by ouabain or omission of bath K⁺ leaves the membrane potential unaffected for a prolonged period or even induces a marked membrane hyperpolarization, although the concentration gradients for K⁺ and Na⁺ are attenuated substantially. As shown previously, this stabilization of the membrane potential is caused by an increase in the K⁺ conductance of the plasma membrane, which compensates for the reduction of the K⁺ gradient. The data presented here strongly suggest that the increased K⁺ conductance is due to Na⁺-activated K⁺ (K_Na) channels. Specifically, an increase in the cytosolic Na⁺ concentration ([Na⁺]_i) was paralleled by a membrane hyperpolarization, a decrease in the input resistance (R_in) of the cells, and by the occurrence of an outwardly directed membrane current. The relationship between R_in and [Na⁺]_i followed a simple model in which the R_in decrease was attributed to K⁺ channels that are activated by the binding of three Na⁺ ions, with half-maximal activation at [Na⁺]_i between 45 and 70 mM. At maximum channel activation, R_in was reduced by more than 90%, suggesting a significant contribution of the K_Na channels to the physiological functioning of the cells, although evidence for such a contribution is still lacking. Injection experiments showed that the K_Na channels in leech P neurons are also activated by Li⁺.     

The Sodium-Potassium Exchange Pump: II. Analysis of Na+-Loaded Frog Sartorius Muscle

A model for the Na-K exchange pump was applied to data on Na⁺-loaded frog sartorius muscle, and was used to relate the rate of adenosine triphosphate (ATP) hydrolysis to the electrical properties of the cell membrane. Membrane hyperpolarization was considered to arise from an electrical current which was produced by the hydrolysis reaction coupled to ion movements, and which flowed across the membrane. The reaction rate, as calculated from hyperpolarization, agreed with direct measurements of ATP hydrolysis and with the rate estimated from Na⁺ tracer efflux studies. Although Na⁺ is actively extruded, the model showed that K⁺ is inwardly transported if the potassium permeability of the membrane is less than about 6.6 × 10^-6 cm/sec, as is suggested by resistance data. Calculations indicated that the reaction conductance L_rr was relatively constant when compared with the reaction rate and reaction free energy for large changes in internal and external ionic concentrations. Its value agreed with the value obtained from the dependence of Na⁺ tracer efflux on external K⁺. A set of experiments was suggested which would provide a more complete interpretation of the data.     

The effects of alterations of transmembrane Na+ and K+ gradients by ionophores (nigericin,monensin) on serotonin transport in human blood platelets

Ionophores (monensin, nigericin) capable of transporting both Na⁺ and K⁺ across cell membranes down their concentration gradients reduce the rate and total magnitude of serotonin uptake by platelets. The effect of the ionophores was time dependent, so that inhibition increased progressively until eventually uptake ceased entirely. Nigericin and monensin produced loss of platelet K⁺ and an equivalent molar uptake of Na⁺ thereby abolishing the normal transmembrane Na⁺ and K⁺ gradients. The time course of these ionophore-induced cation shifts at 37° C corresponded to the rate at which inhibition of serotonin transport developed. The ionophores did not affect total ATP concentration of platelets nor the metabolic pool of ATP formed from [¹⁴C] adenine. Nigericin and monensin released about 80% of platelet ¹⁴C and endogenous serotonin over a 30 min period, without release of platelet adenine nucleotides, calcium or β-glucuronidase. The ionophores did not elicit platelet aggregation nor did they prevent maximal aggregation brought about by ADP, collagen or A23187. Replacement of Na⁺ in the medium by K⁺ abolished serotonin uptake but only 10–20% of endogenous serotonin was released. In KCl medium the Na⁺ gradient was initially reversed, but quickly dissipated as Na⁺ reequilibrated with the extracellular fluid. At 37° C the ionophores did not affect either the rate of Na⁺ reequilibration or the efflux of [¹⁴C] serotonin. Na⁺ reequilibration was slower at 20° C and the ionophores significantly increased platelet Na⁺ loss and strongly inhibited the efflux of [¹⁴C] serotonin. The data support a mechanism of serotonin transport due to a Na⁺-dependent carrier-mediated process which need not be directly dependent on metabolic energy, but which does require metabolic energy to maintain normal $\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$ gradients.     

Characterization of the carrier-mediated [3H]GABA release from isolated synaptic plasma membrane vesicles

Synaptic plasma membrane (SPM) vesicles were isolated under conditions which preserve most of their biochemical properties. Therefore, they appeared particularly useful to study the cytoplasmic GABA release mechanism through its neuronal transporter without interference of the exocytotic mechanism. In this work, we utilized SPM vesicles isolated from sheep brain cortex to investigate the process of [³H]GABA release induced by ouabain, veratridine and Na⁺ substitution by other monovalent cations (K⁺, Rb⁺, Li⁺, and choline). We observed that ouabain is unable to release [³H]GABA previously accumulated in the vesicles and, in our experimental conditions, it does not act as a depolarizing agent. In contrast, synaptic plasma membrane vesicles release [³H]GABA when veratridine is present in the external medium, and this process is sensitive to extravesicular Na⁺ and it is inhibited by extravesicular Ca²⁺ (1 mM) under conditions which appear to permit its entry. However, veratridine-induced [³H]GABA release does not require membrane depolarization, since this drug does not induce any significant alteration in the membrane potential, which is determined by the magnitude of the ionic gradients artificially imposed to the vesicles. The substitution of Na⁺ by other monovalent cations promotes [³H]GABA release by altering the Na⁺ concentration gradient and the membrane potential of SPM vesicles. In the case of choline and Li⁺, we observed that the fraction of [³H]GABA released relatively to the total amount of neurotransmitter released by K⁺ or Rb⁺ is about 28% and 68%, respectively. Since the replacement of Na⁺ by K⁺, Rb⁺, and Li⁺ causes different levels of membrane depolarization, and the replacement of Na⁺ by choline causes hyperpolarization of the vesicles, these results suggest that, in parallel to the [³H]GABA release, which is directly proportional to the level of membrane depolarization, this neurotransmitter can be released by decreasing the external Na⁺, which reflects an elevation of the Na⁺ concentration gradient (inout). Like veratridine-induced release, the depolarization-induced release of [³H]GABA by SPM vesicles is inhibited by Ca²⁺, which suggests that this divalent cation interfers with the cytoplasmic GABA release mechanism.Abbreviations used ATPase adenosine triphosphatase - GABA -aminobutyric acid - Mes 2 (N-morpholino)-ethanosulfonic acid - SPM synaptic plasma membranes - membrane potential     

CHARACTERIZATION OF MEMBRANES OBTAINED FROM ELECTRIC ORGAN OF THE ELECTRIC EEL BY SUCROSE GRADIENT FRACTIONATION AND BY MICRODISSECTION

Abstract— Two membrane fractions were obtained from electric organ tissue of the electric eel by sucrose gradient centrifugation of tissue homogenates. Electron microscopic examination showed that both fractions contained mainly vesicular structures (microsacs). Both the light and heavy fractions had a-bungarotoxin-binding capacity and Na⁺-K⁺ ATPase activity, while only the light fraction had AChE activity. The polypeptide patterns of vesicles derived from both the light and heavy fractions were examined by SDS-polyacrylamide gel electrophoresis and found to be very similar. The ratio of protein to phospholipid in the light vesicles was much lower than in the heavy vesicles, but the relative amounts of individual phospholipids in the two fractions were similar. A marked difference in the permeability of the light and heavy vesicles was observed by measuring efflux of both [¹⁴C]sucrose and ²²Na⁺, and also by monitoring volume changes induced by changing the osmotic strength of the medium. All three methods showed the heavy vesicles to be much more permeable than the light ones. Only the light vesicles displayed increased sodium efflux in the presence of carbamylcholine. The AChE in the light fraction does not appear to be membrane-bound, but is rather a soluble enzyme, detached from the membrane during homogenization, which migrates on the gradient similarly to that of the light vesicles. This is supported by the fact that the bulk of the AChE is readily removed by washing the vesicles. Moreover, under the conditions employed in our sucrose gradient separations,‘native’14 S + 18 S AChE exists in the form of aggregates which migrate very similarly to the major peak of AChE activity of tissue homogenates. Separated innervated and non-innervated surfaces of isolated electroplax were obtained by microdissection. α-Bungarotoxin-binding capacity was observed only in the innervated membrane. About 80% of the AChE was in the innervated membrane, and about 70% of the Na⁺-K⁺ ATPase in the non-innervated membrane. The data presented indicate that the light and heavy vesicle fractions separated by sucrose gradient centrifugation are not derived exclusively from the innervated and non-innervated membranes respectively, as previously suggested by others, but contain membrane fragments from both sides of the electroplax. The separation of two populations on sucrose gradients may be explained both by the differences in permeability and in protein to phospholipid ratios.     

Cationic interactions with Na+-H+ exchange and passive Na+ flux in cardiac sarcolemmal vesicles

Na⁺-H⁺ exchange and passive Na⁺ flux were investigated in cardiac sarcolemmal vesicles as a function of changing the ionic composition of the reaction media. The inclusion of EGTA in the reaction medium resulted in a potent stumulation of Na⁺ uptake by Na⁺-H⁺ exchange. It was found that millimolar concentrations of Mg²⁺ and Li⁺ were capable of inhibiting Na⁺-H⁺ exchange by 80%. One mechanism by which these ions may inhibit intravesicular Na⁺ accumulation by Na⁺-H⁺ exchange is via an increase in Na⁺ efflux. An examination of Na⁺ efflux kinetics from vesicles pre-loaded with Na⁺ revealed that Na⁺, Ca²⁺, Mg²⁺ and Li⁺ could stimulate Na⁺ efflux. Na⁺-H⁺ exchange was potently inhibited by an organic divalent cation, dimenthonium, which screens membrane surface charge. This would suggest that Na⁺-H⁺ exchange occurs in the diffuse double layer region of cardiac sarcolemma and this phenomenon is distinctly different from other Na⁺ transport processes. The results in this study indicate that in addition to a stimulation of Na⁺ efflux, the inhibitory effects of Mg²⁺, Ca²⁺ and Li⁺ on Na⁺-H⁺ exchange may also involve a charge dependent screening of Na⁺ interactions with the membrane.     

Multipurpose Na+ ions mediate excitation and cellular homeostasis: Evolution of the concept of Na+ pumps and Na+/Ca2+ exchangers

Ionic signalling is the most ancient form of regulation of cellular functions in response to environmental challenges. Signals, mediated by Na⁺ fluxes and spatio-temporal fluctuations of Na⁺ concentration in cellular organelles and cellular compartments contribute to the most fundamental cellular processes such as membrane excitability and energy production. At the very core of ionic signalling lies the Na⁺-K⁺ ATP-driven pump (or NKA) which creates trans-plasmalemmal ion gradients that sustain ionic fluxes through ion channels and numerous Na⁺-dependent transporters that maintain cellular and tissue homeostasis. Here we present a brief account of the history of research into NKA, Na⁺ -dependent transporters and Na⁺ signalling.     

Sidedness of the ATP-Na+-K+ interactions with the Na+ pump in squid axons

Using dialysed squid axons we have been able to control internal and external ionic compositions under conditions in which most of the Na⁺ efflux goes through the Na⁺ pump. We found that (i) internal K⁺ had a strong inhibitory effect on Na⁺ efflux; this effect was antagonized by ATP, with low affinity, and by internal Na⁺, (ii) a reduction in ATP levels from 3 mM to 50 μM greatly increased the apparent affinity for external K⁺, but reduced its effectiveness compared with other monovalent cations, as an activator of Na⁺ efflux, and (iii) the relative effectiveness of different K⁺ congeners as external activator of the Na⁺ efflux, though affected by the ATP concentration, was not affected by the Na⁺/⁺ ratio inside the cells. These results are consistent with the idea that the same conformation of the (Na⁺ + K⁺)-ATPase can be reached by interaction with external K⁺ after phosphorylation and with internal K⁺ before rephosphorylation. They also stress a nonphosphorylating regulatory role of ATP.     

Effect of K+ and K+ gradients on accumulation of sugars by isolated intestinal epithelial cells

Summary The role played by transmembrane K⁺ gradients in providing an energy input for Na⁺-dependent monosaccharide transport systems was evaluated with the use of isolated intestinal epithelial cells. Experimentally imposing a K⁺ gradient in a sense reversed from normal did not lead to extrusion of sugar from cells which had been pre-equilibrated with¹⁴C-3-OMG, even in situations where a reversed Na⁺ gradient was also imposed. Furthermore, cells preloaded with K⁺ have no better ability to accumulate 3-OMG than do cells depleted of K⁺, when the two populations are compared under identical incubation conditions. Fluxes of K⁺ associated with the sugar carrier could not be detected in terms of suspected sensitivity to agents which immobilize the sugar carrier. In addition, fluxes of sugar in response to imposed K⁺ gradients were not demonstrable in cells de-energized by preincubation with DNP, no matter in which direction the K⁺ gradient was imposed. Finally, the severe inhibitory effects of K⁺ on Na⁺-dependent sugar transport by the cells disappears in de-energized cells, despite the fact that Na⁺-dependent carrier-mediated sugar entry still occurs. All of these facts are difficult to reconcile with a significant role for cellular K⁺ gradients in supporting active sugar transport as envisioned by the ion gradient hypothesis. We have suggested instead a fundamental Na⁺-dependent energy transductive event which depends on ATP, and which can generate a membrane-bound energized intermediate which serves to support a variety of active transport events. An analogy is drawn between this concept for animal cell plasma membranes and the better documented phosphotransferase system for sugar transport described for certain microorganisms.     

Amiloride-sensitive Na+ transport in human red cells: Evidence for a Na/H exchange system

Summary The role of transmembrane pH gradients on the ouabain, bumetanide and phloretin-resistant Na⁺ transport was studied in human red cells. Proton equilibration through the Jacobs-Stewart cycle was inhibited by the use of DIDS (125 m) and methazolamide (400 m). Red cells with different internal pH (pH _i =6.4, 7.0 and 7.8) were prepared and Na⁺ influx was measured at different external pH (pH _o =6.0, 7.0, 8.0). Na⁺ influx into acid-loaded cells (pH _i =6.4) markedly increased when pH _o was raised from 6.0 to 8.0. Amiloride, a well-known inhibitor of Na⁺/H⁺ exchange systems blocked about 60% of the H⁺-induced Na⁺ entry, while showing small inhibitory effects in the absence of pH gradients. When pH₀ was kept at 8.0, the amiloride-sensitive Na⁺ entry was abolished as pH _i was increased from 6.4 to 7.8. Moreover, measurements of H⁺ efflux into lightly buffered media indicated that the imposition of an inward Na⁺ gradient stimulated a net H⁺ efflux which was sensitive to the amiloride analog 5-N-methyl-N-butyl-amiloride. Furthermore, in the absence of a chemical gradient for Na⁺ (Na _i ⁺ =Na ₀ ⁺ =15mm,Em=+6.7 mV), an outward H⁺ gradient (pH _i =6.4, pH₀=8.0) promoted a net amiloride-sensitive Na⁺ uptake which was abolished at an external pH of 6.0. These findings are consistent with the presence of an amiloride-sensitive Na⁺/H⁺ exchange system in human red cells.     

Plasma membrane potential of Lettré cells does not depend on cation gradients but on pumps

Summary The plasma membrane potential of Lettré cells has been determined with the optical indicator oxonol-V and found to be –57 mV at 37°C (range –20 to –80 mV depending on the physiological condition of the cells). Increasing extracellular K⁺ does not depolarize cells: even in the presence of 155mM K⁺ the potential is –41 mV; membrane potential is also insensitive to the chemical gradient of Na⁺,Mg²⁺, Ca²⁺ or Cl^–. Ouabain depolarizes the cells; H⁺ efflux from cells is stimulated by extracellular Na⁺. We propose that in Lettré cells the plasma membrane potential is generated by electrogenic cation pumps. The balancing fluxes of Na⁺ and K⁺ are mainly through electroneutral cation exchanges (Na⁺/K⁺ and Na⁺/H⁺) and the magnitude of the potential is limited by organic anion leaks. Such a mechanism may operate in other biological membranes also.     

The role of calcium in salt toxicity

Salt toxicity comprises osmotic and ionic components both of which can severely affect root and shoot growth. Uptake of Na⁺ across the plasma membrane is very fast resulting in physiological effects on extracellular as well as intracellular sites. Sodium reduces binding of Ca²⁺ to the plasma membrane, inhibits influx while increasing efflux of Ca²⁺, and depletes the internal stores of Ca²⁺ from endomembranes. These changes in the cell Ca²⁺ homeostasis are suggested here to be the primary responses to salt stress that are perceived by root cells. Salt would almost instantly reduce the amount of Ca²⁺ being transferred to the leaf cells, with Ca²⁺ activity dropping and Na⁺ activity rising in the apoplasm of leaf cells. This Ca²⁺ signal would be transported to leaves together with, if not preceding, the signal of limited water supply. Hormonal signals are likely to be secondary in nature and caused by the Na⁺-related disturbance of the root cell Ca²⁺ homeostasis. Ameliorative effects of supplemental Ca²⁺ on salt stress are exerted through preventing Na⁺-related changes in the cell Ca²⁺ homeostasis.