Reversible stimulation of the Na+/K+ pump by monensin in cultures of vascular smooth muscle

Two ionophores, monensin and salinomycin, increased total cell Na⁺ and ouabain-sensitive ⁸⁶Rb⁺ uptake in cultures of smooth muscle cells from rat aorta. Monensin was used to produced graded increases in cell Na⁺ in order to assess the Na⁺ dependence of the Na⁺/K⁺ pump in the intact cell. The relationship between internal Na⁺ and ouabain-sensitive ⁸⁶Rb⁺ uptake was hyperbolic (K¹_{Na = 3 mM)}. Monensin did not stimulate ⁸⁶Rb⁺ uptake in the absence of external Na⁺. Loading the cells with Na⁺ by exposing cultures to a K⁺-free medium for 3 hr maximally increased cell Na⁺ and ouabain-sensitive ⁸⁶Rb⁺ uptake to the same extent as monensin. Total cell Na⁺ and pump activity in monensin-treated cells returned to the initial values after removing the ionophore. Monensin was then able to increase total cell Na⁺ and ouabain-sensitive ⁸⁶Rb⁺ uptake to the same extent as the initial treatment with the ionophore.     

Sodium-pump activity and its inhibition by extracellular calcium in cardiac myocytes of guinea pigs

Myocardial sodium-pump activity was examined from ouabain-sensitive 86Rb+ uptake using myocytes isolated from guinea-pig heart. Either sodium loading or the sodium ionophore, monensin, increased 86Rb+ uptake by over 400%, indicating that the amount of Na+ available to the pump is the primary determinant of its activity, and that the sodium pump has a substantial reserve capacity in quiescent myocytes. Moreover, the degree of the above stimulation is markedly higher than corresponding values reported with multicellular preparations, suggesting that diffusion barriers make it impossible to observe the capacity of the sodium pump in the latter preparations. Removal of extracellular Ca2+ increased ouabain-sensitive 86Rb+ uptake, probably by enhancing turnover of the sodium pump rather than increasing availability of Na+ to the pump.     

Insulin action on cardiac glucose transport Studies on the role of the Na+/K+ pump

Isolated muscle cells from adult rat heart have been used to study the relationship between myocardial glucose transport and the activity of the Na⁺/K⁺ pump. ⁸⁶Rb⁺-uptake by cardiac cells was found to be linear up to 2 min with a steady-state reached by 40–60 min, and was used to monitor the activity of the Na⁺/K⁺ pump. Ouabain (10^?3 mol/I) inhibited the steady-state uptake of ⁸⁶Rb⁺ by more than 90%. Both, the ouabain-sensitive and ouabain-insensitive ⁸⁶Rb⁺-uptake by cardiac cells were found to be unaffected by insulin treatment under conditions where a significant stimulation of 3-O-methylglucose transport occurred. ⁸⁶Rb⁺-uptake was markedly reduced by the presence of calcium and/or magnesium, but remained unresponsive towards insulin treatment. Inhibition of the Na⁺/K⁺ pump activity by ouabain and a concomitant shift in the intracellular Na⁺:K⁺ ratio did not affect basal or insulin stimulated rates of 3-O-methylglucose transport in cardiac myocytes. The data argue against a functional relationship between the myocardial Na⁺/K⁺ pump and the glucose transport system.     

Errata

1. (1) The significance of the specific (ouabain-sensitive) ⁸⁶Rb⁺ or ⁴²K⁺ uptake by cardiac muscle preparations which are not ‘sodium-loaded’ was studied.

2. (2) In left atrial preparations of guinea-pig heart, resting ⁸⁶Rb⁺ uptake was relatively low. It was markedly increased by electrical stimulation. This stimulated uptake was further enhanced by isoproterenol and inhibited by verapamil.

3. (3) In rat atria, the resting ⁸⁶Rb⁺ uptake was somewhat higher than in guinea-pig atria, and the increase in uptake caused by electrical stimulation was smaller. In guinea-pig right ventricular papillary muscle, the resting uptake was highest among those tissues studied, and the response to electrical stimulation was smallest. In the latter tissue, verapamil produced only a minimal inhibition of the specific ⁸⁶Rb⁺ uptake.

4. (4) The effect of the frequency of electrical stimulation on ⁸⁶Rb⁺ uptake paralleled its influence on the force of contraction, suggesting the involvement of intracellular sodium in both events.

5. (5) In both left atrial and right papillary muscle preparations of guinea-pig heart, specific ⁴²K⁺ uptake observed with 5.8 mM K⁺ was relatively high, and was increased only slightly by electrical stimulation. This electrical stimulation, however, increased ouabain-induced inhibition of ⁴²K⁺ uptake, suggesting that the stimulation increases the amount of Na⁺ available to the sodium pump.

6. (6) When the K⁺ concentration was 1 mM, the resting ⁴²K⁺ uptake was low, and could be enhanced by electrical stimulation.

Stretch-induced growth of skeletal myotubes correlates with activation of the sodium pump

Skeletal myotubes responded to passive stretch by increased amino acid uptake (as measured with [³H]α-aminoisobutyric acid), increased incorporation of amino acids into total cellular protein and myosin heavy chains, and increased accumulation of total cellular protein and myosin heavy chains. These alterations were preceded by an increase in the uptake of ouabain-sensitive rubidium-86 (⁸⁶Rb⁺), a potassium tracer used to measure membrane sodium pump activity (Na⁺K⁺ATPase). This stretch-induced stimulation of ⁸⁶Rb⁺ uptake resulted from a 60-70% increase in the V_max of the Na pump with little change in the K_m. [³H] ouabain binding studies showed no stretch-induced change in the number of membrane Na pumps, indicating that stretch activates the Na pumps that are already present on the cell surface. Since the stretch-induced increases in amino acid transport and amino acid incorporation into proteins were inhibited by ouabain, Na pump activation may be involved in stretch-induced cell growth of skeletal muscle cells by hypertrophy.     

Regulation of Na+, K+-ATPase activity in MDCK kidney epithelial cell cultures: Role of growth state,cyclic AMP,and chemical inducers of dome formation and differentiation

Na⁺,K⁺-ATPase activity was monitored in MDCK kidney epithelial cell monolayers and in cell extracts as a function of cell density, cAMP elevation, and exposure to hexamethylene bisacetamide (HMBA) and dimethylsulfoxide (Me₂SO). Ouabain-sensitive Na⁺,K⁺-ATPase and ⁸⁶Rb⁺ uptake activities, and the number of [³H]-ouabain binding sites were maximal in subconfluent cultures and decreased accompanying the development of a confluent monolayer. A sodium pump density of 8 × 10⁷ pumps/cell was estimated for subconfluent cultures, declining to 9 × 10⁵ pumps/cell at confluence. Previous studies have shown that dibutyryl cyclic AMP (Bt₂cAMP), 1-methyl-3-isobutylxanthine (IBMX), or the differentiation inducers HMBA and Me₂SO, which also caused cAMP elevation, all stimulated dome formation, a visible manifestation of active transepithelial Na⁺ and water transport (Lever, 1979). In the present study, all of these inducers were found to elevate intracellular Na⁺ content, implicating this variable in control of induction of dome formation. Operationally, inducers could be divided into two classes. HMBA and Me₂SO partially inhibited ouabain-sensitive ⁸⁶Rb⁺ influx. Ouabain, at a concentration that caused partial sodium pump inhibition and increased intracellular Na⁺ content, was also effective as an inducer. The second class, exemplified by IBMX and Bt₂cAMP caused a furosemide-sensitive increase in intracellular Na⁺ content. This class of inducers stimulated ouabain-sensitive ⁸⁶Rb⁺ uptake, presumably by substrate effects due to increased Na⁺ levels. The Na⁺ or ATP activation of Na⁺,K⁺-ATPase activity assayed in cell-free extracts, the affinity of the transport system for Rb⁺ in intact cells and intracellular ATP levels were unchanged by inducer treatment. Elevation of intracellular Na⁺ concentration, either by cAMP-stimulated, furosemide-sensitive mechanisms or by partial inhibition of the sodium pump may stimulate the induction of dome formation in MDCK cells.     

Modulation of β-Cell Ouabain-Sensitive 86Rb+ Influx (Na+/K+ Pump) by D-Glucose,Glibenclamide or Diazoxide

The activity of the β-cell Na⁺/K⁺ pump was studied by using ouabain-sensitive (lmM ouabain) ⁸⁶Rb⁺ influx in β-cell-rich islets of Umeå-ob/ob mice as an indicator of the pump function. The present results show that the stimulatory effect of glucose on ouabain-sensitive ⁸⁶Rb⁺ influx reached its approximate maximum at 5mM glucose. Pre-treatment of the islets with 20mM glucose for 60 min strongly reduced the glucose-induced stimulation of the Na⁺/K⁺ pump. Pre-treatment (60 or 180 min) of islets at 0mM glucose, on the other hand, did not affect the magnitude of the glucose-induced stimulation of ⁸⁶Rb⁺ influx dunng the subsequent 5-min incubation. Glibenclamide stimulated the ouabain-sensitive ⁸⁶Rb⁺ uptake in the same manner as glucose. The stimulatory effect, showed its apparent maximum at 0.5μM. Pre-treatment (60 min) of islets with 1μM glibenclamide did not reduce the subsequent stimulation of the ouabain-sensitive ⁸⁶Rb⁺ influx. The stimulatory effect of glibenclamide and D-glucose were not .additive, suggesting that they may have the same mechanism of action. No direct effect of glibenclamide (0.01-1μM) was observed on the Na⁺/K⁺ ATPase activity in homogenates of islets. Diazoxide (0.4mM) inhibited the Na⁺/K⁺ pump. This effect was sustained even after 60 min of pre-treatment of islets with 0.4mM diazoxide. The stimulatory effect of glibenclamide and D-glucose were abolished by diazoxide. It is concluded that nutrient as well as non-nutrient insulin secretagogues activate the Na⁺/K⁺ pump, probably as part of the membrane repolarisation process.     

Cell growth and ouabain-sensitive Rb uptake and (Na + K)-ATPase activity in 3T3 and SV40 transformed 3T3 fibroblasts

The uptake of ouabain-sensitive ⁸⁶Rb⁺ uptake measured at 5 min and the uptake measured at 60 min was 4.5- and 2.7-fold greater respectively for SV40 transformed 3T3 cells compared to 3T3 cells during the late log phase of growth. This uptake, however, varied markedly with cell growth. Ouabain-sensitive ⁸⁶Rb⁺ uptake was found to be a sensitive indicator of protein synthesis as measured by total protein content. Cessation of cell growth as measured by total protein content was associated with a decline in ouabain-sensitive ⁸⁶Rb⁺ uptake in both cell types. This increased ouabain-sensitive cation transport was reflected in increased levels of (Na⁺ + K⁺)-ATPase activity for SV40 3T3 cells, which showed a 2.5-fold increase V but the same K_rmm as 3T3 cells.These results are compared with the results of related work. Possible mechanisms for these effects are discussed and how changes in cation transport might be related to alterations in cell growth.     

Influence of Bcl-2 overexpression on Na+/K+-ATPase pump activity: Correlation with radiation-induced programmed cell death

Bcl-2 overexpression in transfected PW cells is associated with inhibition of radiation-induced programmed cell death (PCD). We have previously reported that there is a relationship between inhibition of radiation-induced PCD and membrane hyperpolarization in these cells. In this article, we report that Na⁺/K⁺-ATPase pump activity, as measured by the uptake of Rubidium-86 (⁸⁶Rb⁺), is significantly higher in Bcl-2 overexpressing PW cells than in control PW cells, and that pump activity following irradiation with doses ≥ 500 cGy was reduced to a lesser extent in the Bcl-2 transfectants than in the control cells. When PW-Bcl-2 cells were incubated with a dose of ouabain (1 μM) that decreased pump activity significantly, but did not induce PCD, the previously reported protection from radiation-induced PCD associated with overexpression of Bcl-2 no longer existed. In order to demonstrate that reactive oxygen species (ROS) affected Na⁺/K⁺-ATPase pump activity, cells were incubated with N-acetyl cysteine (NAC) prior to irradiation, or treated with the ROS generating drug buthionine sulphoxamine (BSO). ⁸⁶Rb⁺uptake was significantly higher in irradiated cells incubated with NAC compared to cells irradiated in the absence of NAC, while BSO resulted in lower levels of ⁸⁶Rb⁺uptake, suggesting that the effects of radiation on the Na⁺/K⁺-ATPase pump were due to ROS. Furthermore, the resting cell membrane potential of cells exposed to NAC were slightly hyperpolarized compared to control PW cells, whereas cells exposed to BSO were depolarized in comparison to control PW cells. In summary, this data suggests that Bcl-2 affects Na⁺/K⁺-ATPase pump activity, which is associated with the resting membrane potential and the level of susceptibility to radiation-induced PCD. J. Cell. Physiol. 171:299–304, 1997. © 1997 Wiley-Liss, Inc.     

Cell Cycle activation and ouabain-sensitive ion movements of 3T3 and C3H-10T½ fibroblasts

The introduction of either PGF_2α (10^?7 M) or TPA (10^?7 M) stimulated, ouabain-sensitive ⁸⁶Rb⁺ influx at 30 min in postconfluent 3T3-4 mouse fibroblast cultures by 117% and 124%, respectively. Both TPA and PGF_2α at these concentrations stimulated the incorporation of ³H-TdR into DNA. TPA had the greatest stimulatory effect, which was similar to that obtained with 10% fetal calf serum. In accord with the idea that modulation of membrane processes such as Na⁺/K⁺ pump activity in fibroblasts may reflect important events related to the initiation of DNA synthesis, it was observed that in both 3T3-4 and C3H-1 0T½ cells there were parallel increases in ³H-TdR incorporation and ouabain-sensitive ⁸⁶Rb⁺ influxes with 10^?7 M TPA, whereas PGF_2α stimulated a significant increase in ³H-TdR incorporation in 3T3-4 but not C3H-10T½ cells and only marginal increases in ouabain-sensitive ⁸⁶Rb⁺ influx in both. Therefore, although there appears to be a close correlation between Na⁺/K⁺ pump activation and subsequent S-phase entry following TPA stimulation, a similar correlation for PGF_2α cannot be confirmed.     

Exogeneous diacylglycerols downregulate the activity of Na-K pump in Xenopus laevis oocytes

In this study, cell permeable diacyglycerols, sn-1,2-dioctanoglycerol (DiC8), and sn-1-oleoyl-2-acetylglycerol (OAG) were found to downregulate the activity of Na⁺-K⁺ pump in Xenopus laevis oocytes. Both DiC8 and OAG decreased the binding of [³H]ouabain to intact oocytes while phorbol esters did not appreciably influence the same. These diacylglycerols inhibited the amiloride-sensitive ²²Na⁺ influx and ouabain-sensitive ⁸⁸Rb⁺ uptake in the oocytes. Furthermore, DiC8 prevented the ²²Na⁺ efflux from the oocytes preloaded with ²²Na⁺. Addition of H-7 to DiC8- and OAG-treated oocytes stimulated the pump activity curtailed by the two latters. The impairment of Na⁺-K⁺ pump activity by diacylglycerols suggests that protein kinase C activators may stimulate endocytosis of membrane-coupled Na⁺-K⁺ ATPase.     

Selective inhibition by bis(2-chloroethyl)methylamine (nitrogen mustard) of the Na/K/Cl cotransporter of murine L1210 leukemia cells

Incubation of L1210 murine leukemia cells in vitro with 10 μM of the bifunctional alkylating agent bis(2-chloroethyl)methylamine (nitrogen mustard, HN2) for 10 min brought about a fall of more than 99.9% in their ability to form colonies when the cells were suspended in 0.5% nutrient agar. Incubation with HN2 also inhibited the influx of the potassium congener ⁸⁶Rb⁺ to exponentially proliferating L1210 cells in a concentration-dependent manner. This inhibition was specific and was accounted for by a reduction of a diuretic-sensitive component of ⁸⁶Rb⁺ influx, identified in the preceding paper (Wilcock, C. and Hickman, J.A. (1988) Biochim. Biophys. Acta 946, 359–367) as being mediated by a Na⁺/K⁺/Cl⁻ cotransporter. Inhibition by 10 μM HN2 was complete after a 3-h incubation. There was no inhibition at this time of the ouabain-sensitive component of ⁸⁶Rb⁺ influx, mediated by Na⁺/K⁺-ATPase. After 3 h of incubation with 10 μM HN2 there was also no change in the membrane potential of the treated cells as measured by the distribution of the [³H]TPMP⁺, no decrease in cellular ATP concentration and no change in intracellular pH, and the ability of the cells to exclude the vital dye Trypan blue was not significantly different from control values. These effects of HN2, therefore, appeared to follow lethal damage, but precede cell death. In the stationary phase of L1210 cell growth, the component of HN2 and diuretic-sensitive K⁺ influx to L1210 cells was reduced, whilst the component constituting the HN2-insensitive ouabain-sensitive sodium pump was increased. The monofunctional alkylating agent MeHN1 (2-chloroethyldimethylamine) which cannot cross-link cellular targets and has no antitumour activity, did not inhibit ⁸⁶Rb⁺ influx to L1210 cells when incubated at equimolar or equitoxic concentrations to HN2. Intracellular potassium concentration was maintained close to control values of 138 ± 10 mM in HN2-treated cells because of an approx. 35% fall in cell volume. The results suggest that the Na⁺/K⁺/Cl⁻ cotransporter is a selectively inhibitable target for HN2, and the lesion is discussed with reference to the cytotoxic effects of this agent.     

Pig reticulocytes. V. Development of Rb+ influx during in vitro maturation

Influx of the K⁺ analogue Rb⁺ was measured through the ouabain-sensitive Na⁺/K⁺ pump and the ouabain-insensitive “leak” pathways in Cl^? or NO in mature red cells from adult pigs and in reticulocytes naturally occurring in 7-day-old piglets. In reticulocytes, Rb⁺ influxes by the two pathways were of about equal magnitude in Cl^? (13 and 10 mmoles/liter cells × hr) and at least 25-fold larger than in mature red cells (0.5 and 0.4 mmoles/liter cells × hr). In Na ⁺ media, a portion of the ouabain-insensitive “leak” flux of Rb⁺ was Cl^? dependent (Rb⁺Cl^? transport) as NO replacement reduced Rb⁺ influx by 90% in reticulocytes and by 40% in mature red cells. The sulfhydryl reagent N-ethylmaleimide (NEM) stimulated Rb⁺Cl^? transport about twofold in reticulocytes and up to 13-fold in mature red cells. When reticulocytes matured to erythrocytes during in vitro incubation, about 90% of both ouabain-sensitive Rb⁺ pump and ouabain-insensitive Rb⁺Cl^? influx were lost. In contrast, the NEM-stimulated Rb⁺Cl^? transport changed much less throughout this period, suggesting an entity operationally but not necessarily structrually distinct from the basal Rb⁺Cl^? transport. Although the experimental variability precluded a full assessment of significant changes in the small Na⁺/K⁺(Rb⁺) pump and Rb⁺Cl^? fluxes in mature pig red cells kept for the same time period in vitro, Rb⁺ flux changes in reticulocytes appear to be maturational in nature, reflecting parallel activity transitions of Na⁺/K⁺ pump and Cl^?-dependent K⁺ fluxes in vivo.     

D-glucose Stimulates the Na+/K+ Pump in Mouse Pancreatic Islet Cells

To determine the effect of D-glucose on the β-cell Na⁺/K⁺ pump, ⁸⁶Rb⁺ influx was studied in isolated, -cell-rich islets of Umeå-ob/ob mice in the absence or presence of lmM ouabain. D-glucose (20 mM) stimulated the ouabain-sensitive portion of ⁸⁶Rb⁺ influx by 65%, whereas the ouabain-resistant portion was inhibited by 48%. The Na⁺/K⁺ ATPase activity in homogenates of islets of Umeå-ob/ob mice or normal mice was determined to search for direct effects of D-glucose. Thus, ouabain-sensitive ATP hydrolysis in islet homogenates was measured in the presence of different D-glucose concentrations. No effect of D-glucose (3–20 mM) was observed in either ob/ob or normal islets at the optimal Na⁺/K⁺ ratio for the enzyme (135 mM Na⁺ and 20 mM K⁺). Neither D-glucose (3–20 mM) nor L-glucose or 3-O-methyl-D-glucose (20 mM) affected the enzyme activity at a high Na⁺/K⁺ ratio (175 mM Na⁺ and 0.7mM K⁺). Diphenylhydantoin (150 μM) decreased the enzyme activity at optimal Na⁺/K⁺ ratio, whereas 50 μM of the drug had no effect. The results suggest that D-glucose induces a net stimulation the Na⁺/K⁺ pump of β-cells in intact islets and that D-glucose does not exert any direct effect on the Na⁺/K⁺ ATPase activity.     

Ouabain-sensitive 86Rb(K) influx is linked to transepithelial Na transport in pig kidney cell line

The pig kidney cell line, LLC-PK₁, exhibits rheogenic d-glucose coupled transepithelial Na⁺ transport that is inhibited by phlorizin. By measuring the difference in initial rates of influx of ⁸⁶Rb⁺ with and without coupled Na⁺ transport, we can demonstrate an ⁸⁶Rb⁺ uptake linked to Na⁺ transport. The simultaneous determination of phlorizin-inhibited Na coupled d-[³H]glucose uptake and ⁸⁶Rb⁺ influx allows calculation of an Na⁺/Rb⁺ stoichiometry that is consistent with an electrogenic Na⁺ for Rb⁺ exchange.     

The Na+,K+,2Cl−-cotransport system in HeLa cells: Aspects of its physiological regulation

We have previously reported on the biochemical properties of a Na⁺,K⁺,2Cl^?-cotransport in HeLa cells and here we deal with aspects of its physiological regulation. Na⁺,K⁺,2Cl^?-cotransport in HeLa cells was studied by ⁸⁶Rb⁺ influx and ⁸⁶Rb⁺/²²Na⁺ efflux measurements. The effects of rat atrial natriuretic peptide (ANP), isoproterenol, and amino acids on ⁸⁶Rb⁺ flux, mediated by the bumet-anide-sensitive Na⁺, K⁺, 2Cl^?-cotransport system and the ouabain-sensitive Na⁺/K⁺-pump, were investigated. ANP reduced bumetanide-sensitive ⁸⁶Rb⁺ influx under isotonic as well as under hypertonic conditions. Similar decrease of bumetanide-sensitive ⁸⁶Rb⁺ influx was observed in the presence of 8-bromo-cGMP, while neither isoproterenol as a β-receptor agonist nor 8-bromo-cAMP-could alter bumetanide-sensitive ⁸⁶Rb⁺ influx. Furthermore, efflux of ⁸⁶Rb⁺ and ²²Na⁺ was greatly reduced in the presence of bumetanide and ANP. Together with our recent findings, showing functionally active, high affinity receptors for ANP on HeLa cells (Kort and Koch, Biochim. Biophys. Res. Commun. 168:148–154, 1990), this study indicates that ANP participates in the regulation of the Na⁺, K⁺, 2Cl^?-cotransport system in HeLa cells. Further measurements revealed that amino acids as present in the growth medium (Joklik's minimal essential medium) and the amino acid derivative α-methyl-aminoisobutyric acid (metAlB, 1 and 5 mM, respectively) also reduced Na⁺, K⁺, 2Cl^?-cotransport-mediated ⁸⁶Rb⁺ uptake and diminished the stimulatory effect of hypertonicity on the cotransporter. In addition, the Na⁺/K⁺-pump was markedly stimulated in the presence of amino acids, while neither ANP and 8-Br-cGMP nor isoproterenol and 8-Br-cAMP had a significant effect on the activity of the Na⁺/K⁺-pump.     

A comparison of the ouabain-sensitive (Na+ + K+)-ATPase of normal and dystrophic skeletal muscle

An NaI-extraction procedure was modified to prepare muscle fiber segments with Mg²⁺-dependent, ouabain-sensitive (Na⁺ + K⁺)-ATPase activity. This enzyme was assayed in preparations of skeletal muscle from normal and dystrophic mice. The ouabain-sensitive (Na⁺ + K⁺)-ATPase activity of dystrophic muscle preparations was found to be significantly lower than that of control preparations.     

Furosemide-sensitive K+ channel in glioma cells but not neuroblastoma cells in culture

A furosemide-sensitive, ouabain-insensitive [⁸⁶Rb⁺] uptake is described in glioma cells in culture which is dependent upon external Na⁺, K⁺, and Cl^? concentrations. This transport activity was also inhibited by bumetanide at 100-fold lower concentrations than furosemide. Furosemide-sensitive swelling of glioma cells is demonstrated and this activity is dependent upon external Na⁺ and K⁺ in a manner similar to [⁸⁶Rb⁺] uptake. This transport activity was not detected in neuroblastoma cells and the possible relevance of these findings to extracellular K⁺ buffering by glia is discussed.     

Alkali cation selectivity of the wheat root high-affinity potassium transporter HKT1

The wheat root high-affinity K⁺ transporter HKT1 functions as a sodium-coupled potassium co-uptake transporter. At toxic millimolar levels of sodium (Na⁺), HKT1 mediates low-affinity Na⁺ uptake while potassium (K⁺) uptake is blocked. In roots, low-affinity Na⁺ uptake and inhibition of K⁺ uptake contribute to Na⁺ toxicity. In the present study, the selectivity among alkali cations of HKT1 expressed in Xenopus oocytes and yeast was investigated under various ionic conditions at steady state. The data show that HKT1 is highly selective for uptake of the two physiologically significant alkali cations, K⁺ and Na⁺ over Rb⁺, Cs⁺ and Li⁺. In addition, Rb⁺ and Cs⁺, and an excess of extracellular K⁺ over Na⁺, are shown to partially reduce or block HKT1-mediated K⁺-Na⁺ uptake. Furthermore, K⁺, Rb⁺ and Cs⁺ also effectively reduce outward currents mediated by HKT1, thereby causing depolarizations. In yeast, HKT1 can produce high-affinity Rb⁺ uptake at approximately 15-fold lower rates than for K⁺. Rb⁺ influx in yeast can be mediated by the ability of the yeast plasma membrane proton pump to balance the 35-fold lower HKT1 conductance for Rb⁺. A model for HKT1 activity is presented involving a high-affinity K⁺ binding site and a high-affinity Na⁺ binding site, and competitive interactions of K⁺, Na⁺ and other alkali cations for binding to these two sites. Possible implications of the presented results for physiological K⁺ and Na⁺ uptake in plants are discussed.