Effects of Extracellular pH on UV-Induced K Efflux from Cultured Rose Cells

Ultraviolet (UV) light causes a specific leakage of K⁺ from cultured rose cells (Rosa damascena). During K⁺ efflux, there is also an increase in extracellular HCO₃⁻ and acidification of the cell interior. We hypothesized that the HCO₃⁻ originated from intracellular hydration of respiratory CO₂ and served as a charge balancing mechanism during K⁺ efflux, the K⁺ and HCO₃⁻ being cotransported out of the cell through specific channels. An alternative hypothesis which would yield similar results would be the countertransport of K⁺ and H⁺. To test these hypotheses, we studied the effect of a range of external pH values (pH 5-9), regulated by various methods (pH-stat, 100 millimolar Tris-Mes buffer, or CO₂ partial pressure), on the UV-induced K⁺ efflux. Both UV-C (<290 nanometers) and UV-B (290-310 nanometers) induced K⁺ efflux with a minimum at about pH 6 to 7, and greater efflux at pH values of 5, 8, and 9. Since pH values of 8 and 9 increased instead of reduced the efflux of K⁺, these data are not consistent with the notion that the efflux of K⁺ is dependent on an influx of H⁺, a process that would be sensitive to external H⁺ concentration. We suggest that the effect of pH on K⁺ efflux may be mediated through the titration of specific K⁺-transporting proteins or channels in the plasma membrane. Since we could not detect the presence of carbonic anhydrase activity in cell extracts, we could not use the location of this enzyme to aid in our interpretation regarding the site of hydration of CO₂.     

Mechanisms by which Li+ stimulates the (Na++K+)-dependent ATPase

The addition of LiCl stimulated the (Na⁺+K⁺)-dependent ATPase activity of a rat brain enzyme preparation. Stimulation was greatest in high Na⁺/low K⁺ media and at low Mg. ATP concentrations. Apparent affinities for Li⁺ were estimated at the α-sites (moderate-affinity sites for K⁺ demonstrable in terms of activation of the associated K⁺-dependent phosphatase reaction), at the β-sites (high-affinity sites for K⁺ demonstrable in terms of activation of the overall ATPase reaction), and at the Na⁺ sites for activation. The relative efficacy of Li⁺ was estimated in terms of the apparent maximal velocity of the phosphatase and ATPase reactions when Li⁺ was substituted for K⁺, and also in terms of the relative effect of Li⁺ on the apparent K_M for Mg· ATP. With these data, and previously determined values for the apparent affinities of K⁺ and Na⁺ at these same sites, quantitative kinetic models for the stimulation were examined. A composite model is required in which Li⁺ stimulates by relieving inhibition due to K⁺ and Na⁺ (i) by competing with K⁺ for the α-sites on the enzyme through which K⁺ decreases the apparent affinity for Mg·ATP and (ii) by competing with Na⁺ at low-affinity inhibitory sites, which may represent the external sites at which Na⁺ is discharged by the membrane NA⁺/K⁺ pump that this enzyme represents. Both these sites of action for Li⁺ would thus lie, in vivo, on the cell exterior.     

Variable affinity of the (Na+ + K+)-dependent adenosine triphosphatase for potassium: Studies using beryllium inactivation

Inactivation of the (Na⁺ + K⁺)-dependent ATPase by 50 μm BeCl₂ occurred during brief incubations in the presence of both Mg²⁺ and K⁺. Inactivation followed, initially, a first-order time course, with rate constants sensitive to the concentration of K⁺ (other components held constant). From these data dissociation constants can be calculated for K⁺ binding to sites controlling inactivation. Comparisons of relative affinities for K⁺ analogs (T1⁺ and NH₄⁺), and of sensitivity to reagents altering K⁺ activation (phlorizin and dimethylsulfoxide) indicate that the same K⁺ sites operate for both Be²⁺ inactivation and enzyme activation. With 3 mm MgCl₂ the dissociation constant, K_D, for K⁺ was 1.4 mm, but decreased 20-fold on addition of both Na⁺ and CTP. Alone, Na⁺ increased the apparent K_D for K⁺, either by direct competition or indirectly from its own site, with a K_D of 7 mm. The data suggest a model for K⁺ transport with K⁺ sites on the outer membrane surface that increase in affinity after formation of the phosphorylated enzyme intermediate, sufficiently to bind K⁺ in a high Na⁺ environment. Translocation may occur by an “oscillating pore” mechanism discharging K⁺ at the inner surface, while leaving demonstrable sites of moderate affinity at the outer end of the pore (which preclude attempts to document low-affinity discharge sites).     

Extracellular potassium controls responsiveness of the noradrenergic cAMP system in the rat retina to fluphenazine

The effects of fluphenazine (FLU) on the noradrenaline (NA) induced cAMP-synthesis in intact rat retinae were studied as a function of extracellular K⁺- and Ca²⁺-ions. Thus NA-induced cAMP levels were measured after incubating intact rat retinae with 50 μM NA in the presence or absence of FLU and in the presence of 1 or 10 mM theophylline. Results were: (1) Experimental condition a: standard NA-responses were measured after incubating retinae at 0.75 mM Ca²⁺, at 10 mM theophylline, at 10 μM FLU and at 2 and 0 mM K⁺. FLU does not affect the NA-response at 2 mM K⁺ significantly; however, it inhibits the NA-response at 0 mM K⁺ in this condition. (2) Experimental condition b: NA-responses were measured after incubating retinae at 0.125 mM Ca²⁺, 10 mM theophylline, 10 μM FLU and at 2 and 0 mM K⁺. At 2 mM K⁺ FLU replaces a Ca²⁺ function probably connected with the synthesis part of the NA-cAMP system and NA-responses in this low Ca²⁺ condition are consequently enhanced by FLU; however, FLU inhibits the NA-response at 0 mM K⁺ in this condition. (3) Experimental condition c: NA-responses were measured after incubating retinae at 0.75 mM Ca²⁺, 1 mM theophylline, 10 μM FLU and at 2 and 0 mM K⁺. At 2 mM K⁺ FLU enhances the NA-response by further inhibition of the degradation part of the NA-cAMP system; FLU inhibits the NA-response at 0 mM K⁺ in this condition. (4) The inhibitions of the NA-responses by FLU at 0 mM K⁺ in all three conditions a, b and c showed an apparent K_m of 1 μM. (5) Low concentrations of K⁺ (0.4–0.8 mM) maintain the property of FLU to enhance the NA-responses at condition b (0.125 mM Ca²⁺) and at condition c (1 mM theophylline). Results suggest that the activation of NA-receptor coupled adenylate cyclases (NA-AC-ases) by NA, resulting in activation of phosphodiesterase activity by the NA-elevated cAMP-levels, is sustained by (a) membraneous factor(s) connected to the NA-receptor. This (these) factor(s) is (are) switched off in the absence of K⁺. Evidence has been presented, that Ca²⁺ and FLU do not have access to this intramembraneous factor-enzyme activating moiety of the NA-cAMP system at 0 mM K⁺. Between 0.4 and 0.8 mM K⁺ the factor-enzyme-NA-receptor complex is still intact.     

(Na+,K+)-ATPase of mammalian brain: Effects of temperature on cation and ATP interactions regulating phosphatase activity

The effects of temperature on interactions between univalent cations or ATP and the p-nitrophenylphosphatase activity associated with brain (Na⁺,K⁺)-ATPase were examined. The apparent affinity for K⁺ activation under conditions favoring the moderate affinity site was temperature dependent, increasing with decreasing temperature. A comparison of univalent cations showed that the negative apparent ΔH and ΔS for cation binding increased with increasing apparent cation affinity. In contrast to the case with the moderate affinity sites, apparent affinity for the high affinity K⁺ site was independent of temperature. As temperature decreased, properties of moderate affinity site binding approached those of the high affinity site. The temperature dependence of ATP inhibition was opposite to that for K⁺ activation, with positive apparent ΔH and ΔS. The apparent ΔH and ΔS for cation binding approached those for the overall conformational change to K⁺-sensitive enzyme as cation affinity increased. These data suggest that E2, the K⁺-sensitive form of (Na⁺,K⁺)-ATPase, is stabilized by forces that require a decrease in entropy, explaining the predominant existence of E1 at physiologic temperatures. A conformational change leading to stabilization of E2 at higher temperatures can be produced by binding of univalent cations to a moderate affinity, presumably intracellular, site. This effect is counteracted by ATP. ATP also appears to alter the selectivity of this site to favor Na⁺ over K⁺ binding.     

UV-Stimulated K Efflux from Rose Cells: Counterion and Inhibitor Studies

Irradiation of a washed suspension of cultured rose (Rosa damascena var. Gloire de Guilan) cells with about 1,680 joules per square meter of short wave ultraviolet (UV) light (254 nanometers) caused K⁺ to appear in the external medium. Short-term tracer (⁸⁶Rb⁺) experiments confirmed the earlier suggestion (Wright, Murphy 1978 Plant Physiol 61: 434-436) that UV increases the efflux of K⁺; there was also a small decrease in influx of K⁺. There was a partial recovery of fluxes from the effects of UV radiation, but no net accumulation of K⁺ within 16 to 18 hours after the irradiation. The K⁺ appearing in the medium was matched by an equivalent amount of HCO₃⁻; it was suggested that HCO₃⁻ was the principal counterion for the K⁺ flux induced by UV. Inhibitors of ATP synthesis (10⁻⁵ molar carbonyl cyanide m-chlorophenyl hydrazone; 0.05 millimolar KCN plus 0.75 millimolar salicylhydroxamic acid) strongly reduced the UV-stimulated K⁺ leakage, suggesting that the leakage was dependent in some way on ATP concentration inside the cells. The UV-induced K⁺ leakage was also dependent on temperature and the presence of Ca²⁺ in the external medium.     

Sensitivity of the (Na+ + K+)-ATPase to state-dependent inhibitors: Effects of digitonin and triton X-100

Treatment of a purified (Na⁺ + K⁺)-ATPase preparation from dog kidney with digitonin reduced enzymatic activity, with the (Na⁺ + K⁺)-ATPase reaction inhibited more than the K⁺-phosphatase reaction that is also catalyzed by this enzyme. Under the usual assay conditions oligomycin inhibits the (Na⁺ + K⁺)-ATPase reaction but not the K⁺-phosphatase reaction; however, treatment with digitonin made the K⁺-phosphatase reaction almost as sensitive to oligomycin as the (Na⁺ + K⁺)-ATPase reaction. The non-ionic detergents, Triton X-100, Lubrol WX and Tween 20, also conferred sensitivity to oligomycin on the K⁺-phosphatase reaction (in the absence of oligomycin all these detergents, unlike digitonin, inhibited the K⁺-phosphatase reaction more than the (Na⁺ + K⁺)-ATPase reaction). Both digitonin and Triton markedly increased the K_0.5 for K⁺ as activator of the K⁺-phosphatase reaction, with little effect on the K_0.5 for K⁺ as activator of the (Na⁺ + K⁺)-ATPase reaction. In contrast, increasing the K_0.5 for K⁺ in the K⁺-phosphatase reaction by treatment of the enzyme with acetic anhydride did not confer sensitivity to oligomycin. Both digitonin and Triton also increased the inhibition of the K⁺-phosphatase reaction by ATP and decreased the inhibition by inorganic phosphate and vanadate. These observations are interpreted as digitonin and Triton favoring the E₁ conformational state of the enzyme (manifested by sensitivity to oligomycin and a greater affinity for ATP at the low-affinity substrate sites), as opposed to the E₂ state (manifested by insensitivity to oligomycin, greater sensitivity to phosphate and vanadate, and a lower K_0.5 for K⁺ in the K⁺-phosphatase reaction). In addition, digitonin blocked activation of the phosphatase reaction by Na⁺ plus CTP. This effect is consistent with digitonin dissociating the catalytic subunits of the enzyme, the interaction of which may be essential for activation by Na⁺ plus nucleotide.     

Potassium-p-nitrophenyl phosphate interactions with (Na+ + K+)-ATPase their relevance to phosphatase activity

The K⁺-dependent p-nitrophenylphosphatase activity catalyzed by purified (Na⁺ + K⁺)-ATPase from pig kidney shows substrate inhibition (K_i about 9.5 mM at 2.1 mM Mg²⁺). Potassium antagonizes and sodium favours this inhibition. In addition, K⁺ reduces the apparent affinity for substrate activation, whereas p-nitrophenyl phosphate reduces the apparent affinity for K⁺ activation. In the absence of Mg²⁺, p-nitrophenyl phosphate, as well as ATP, accelerates the release of Rb⁺ from the Rb⁺ occluded unphosphorylated enzyme. With no Mg²⁺ and with 0.5 mM KCl, trypsin inactivation of (Na⁺ + K⁺)-ATPase as a function of time follows a single exponential but is transformed into a double exponential when 1 mM ATP or 5 mM p-nitrophenyl phosphate are also present. In the presence of 3 mM MgCl₂, 5 mM p-nitrophenyl phosphate and without KCl the trypsin inactivation pattern is that described for the E₁ enzyme form; the addition of 10 mM KCl changes the pattern which, after about 6 min delay, follows a single exponential. These results suggest that (i) the shifting of the enzyme toward the E₁ state is the basis for substrate inhibition of the p-nitrophenulphosphatase acitivy of (Na⁺ + K⁺)-ATPase, and (ii) the substrate site during phosphatase activity is distinct from the low-affinity ATP site.     

Stability of the Shab K+ channel conductance in 0 K+ solutions: the role of the membrane potential

Shab channels are fairly stable with K⁺ present on only one side of the membrane. However, on exposure to 0 K⁺ solutions on both sides of the membrane, the Shab K⁺ conductance (G_K) irreversibly drops while the channels are maintained undisturbed at the holding potential. Herein it is reported that the drop of G_K follows first-order kinetics, with a voltage-dependent decay rate r. Hyperpolarized potentials drastically inhibit the drop of G_K. The G_K drop at negative potentials cannot be explained by a shift in the voltage dependence of activation. At depolarized potentials, where the channels undergo a slow inactivation process, G_K drops in 0 K⁺ with rates slower than those predicted based on the behavior of r at negative potentials, endowing the r-V_m relationship with a maximum. Regardless of voltage, r is very small compared with the rate of ion permeation. Observations support the hypothesized presence of a stabilizing K⁺ site (or sites) located either within the pore itself or in its external vestibule, at an inactivation-sensitive location. It is argued that part of the G_K stabilization achieved at hyperpolarized potentials could be the result of a conformational change in the pore itself.     

The effect of pressure and the volume of activation on the monovalent cation and glucose permeabilities of liposomes of varying composition

Measurement of the cation permeability of phospholipid microvesicles as function of pressure confirmed that a single rate-determining step occured in each case.The volume of activation was 20 ml · mole^?1 for Na⁺ and K⁺, and about 40 ml · mole^?1 for valinomycin-mediated K⁺ permeability. It was virtually independent of membrane composition. The results were explained in terms of Träuble's theory of kink-substrate dissociation at the membrane interface involving possible 2g 1 and 2g 2 kink isomers.The volume of activation for d-glucose was 37 ml · mole^?1, which was not significantly different from that for any of the valinomycin-mediated K⁺ permeabilities. However, other data suggest that the rate-limiting steps for the sugar and cation permeabilities are not the same.     

The Hypersensitive Reaction of Tobacco to Pseudomonas syringae pv. pisi: Activation of a Plasmalemma K/H Exchange Mechanism

Net electrolyte efflux from suspension-cultured tobacco cells undergoing the hypersensitive reaction to Pseudomonas syringae pv. pisi resulted from a specific efflux of K⁺ which was accompanied by an equimolar net influx of H⁺. These fluxes began 60 to 90 minutes after inoculation of tobacco cells with bacteria, reached maximum rates of 6 to 9 micromoles per gram fresh weight tobacco cells per hour within 2.5 to 3 hours, and dropped below 4 micromoles per gram per hour within 5 hours. Tobacco cells lost approximately 35% of total K⁺ during this period, and average cellular pH declined by approximately 0.75 pH unit. These events were accompanied by a 30% decrease in cellular ATP. K⁺ and H⁺ fluxes were inhibited by the protonophore (p-trifluoromethoxy)carbonyl cyanide phenylhydrazone and by increasing the K⁺ concentration of the external solution. Tobacco leaf discs inoculated with the bacterium also exhibited a specific net K⁺ efflux and H⁺ influx. These results suggest that induction of the hypersensitive reaction in tobacco proceeds through the activation of a passive plasmalemma K⁺/H⁺ exchange mechanism. It is hypothesized that activation of this exchange is a major contributing factor in hypersensitive plant cell death.     

Interaction of Tetraethylammonium Ion Derivatives with the Potassium Channels of Giant Axons

A number of compounds related to TEA⁺ (tetraethylammoniumion) were injected into squid axons and their effects on g_K (the potassium conductance) were determined. In most of these ions a quaternary nitrogen is surrounded by three ethyl groups and a fourth group that is very hydrophobic. Several of the ions cause inactivation of g_K, a type of ionic gating that is not normally seen in squid axon; i.e., after depolarization g_K increases and then spontaneously decreases to a small fraction of its peak value even though the depolarization is maintained. Observations on the mechanism of this gating show that (a) QA (quaternary ammonium) ions only enter K⁺ channels that have open activation gates (the normal permeability gates). (b) The activation gates of QA-occluded channels do not close readily. (c) Hyperpolarization helps to clear QA ions from the channels. (d) Raising the external K⁺ concentration also helps to clear QA ions from the channels. Observations (c) and (d) strongly suggest that K⁺ ions traverse the membrane by way of pores, and they cannot be explained by the usual type of carrier model. The data suggest that a K⁺ pore has two distinct parts: a wide inner mouth that can accept a hydrated K⁺ ion or a TEA⁺-like ion, and a narrower portion that can accept a dehydrated or partially dehydrated K⁺ ion, but not TEA⁺.     

Alteration of cellular ionic constituents by external ionic conditions,and its significance in the development ofDictyostelium discoideum

Effects of external ionic conditions ofD. discoideum cells were examined in relation to intracellular ionic concentrations, the activity of pyruvate kinase and the amount of ATP. Main components of metal cations in heat extracts of vegetative cells were K⁺, Na⁺, Mg²⁺ and Ca²⁺ whose concentrations in a cell were about 35.0, 3.6, 10.6 and 2.3 mM, respectively. External Na⁺ at the concentration more than 50 mM inhibited the formation of cell aggregates in the presence of 10^?4M Ca²⁺. Such an inhibitory effect of Na⁺ was completely nullified by the addition of more than 10 mM K⁺. External Na⁺ caused a rapid decrease in intracellular K⁺, but an increase in intracellular Na⁺. Furthermore, it was found that the cells containing a high concentration of Na⁺ can develop normally in the presence of exogenous 10 mM K⁺, where intracellular K⁺ was maintaned at about 30 mM, irrespective of a high concentration of intracellular Na⁺ (about 30 mM). These suggest that the Na⁺-inhibition of the development is caused by a decrease in intracellular K⁺, but not by an increase in intracellular Na⁺. Pyruvate kinase extracted from the organism required K⁺ for its activation. The vegetative cells incubated in 50 mM Na⁺ contained only about 10 mM K⁺ which is insufficient for the enzyme activation. However, the amount of ATP in the cells containing less K⁺ was similar to that in those with much K⁺. These results are discussed in relation to the activity of glycolysis.     

Passive potassium ion permeability of Halobacterium halobium cell envelope membranes

Cell envelope vesicles, prepared from Halobacterium halobium, were loaded with 3 M KCl suspended in 3 M NaCl, and the loss of K⁺ was followed at various temperatures. The Arrhenius plot of the K⁺-efflux rates shows a break at 30°C, with higher energy of activation above the break. This temperature dependence is consistent with earlier studies of chain motions in liposomes prepared from isolated lipids. The efflux of K⁺ is more rapid with increasing pH between pH 5 and 7. Since these vesicles do not respire under the experimental conditions it was expected that the K⁺-efflux data would be related to the passive permeability of the membranes to K⁺. The apparent K⁺ permeability at 30°C is 1–2· 10^?10 cm·^?1. This value corresponds to a 5-h half-life for retained K⁺ in the envelope vesicles and to a probably much longer half-life in whole cells. The previously observed ability of Halobacterium to retain K⁺ in the absence of metabolism can thus be explained solely by the permeability characteristics of the membranes.     

Influence of ATP-dependent K<Superscript>+</Superscript>-channel opener on K<Superscript>+</Superscript>-cycle and oxygen consumption in rat liver mitochondria

The influence of the K_ATP⁺-channel opener diazoxide on the K⁺ cycle and oxygen consumption has been studied in rat liver mitochondria. It was found that diazoxide activates the K_ATP⁺-channel in the range of nanomolar concentrations (50–300 nM, K _1/2 ∼ 140 nM), which results in activation of K⁺/H⁺ exchange in mitochondria. The latter, in turn, accelerates mitochondrial respiration in respiratory state 2. The contribution of K_ATP⁺-channel to the mitochondrial potassium cycle was estimated using the selective K_ATP⁺-channel blocker glibenclamide. The data show that the relative contribution of K_ATP⁺-channel in the potassium cycle of mitochondria is variable and increases only with the decrease in the ATP-independent component of K⁺ uptake. Possible mechanisms underlying the observed phenomena are discussed. The experimental results more fully elucidate the role of K_ATP⁺-channel in the regulation of mitochondrial functions, especially under pathological conditions accompanied by impairment of the mitochondrial energy state.     

AtNHX5 and AtNHX6 Control Cellular K+ and pH Homeostasis in Arabidopsis: Three Conserved Acidic Residues Are Essential for K+ Transport

AtNHX5 and AtNHX6, the endosomal Na⁺,K⁺/H⁺ antiporters in Arabidopsis, play an important role in plant growth and development. However, their function in K⁺ and pH homeostasis remains unclear. In this report, we characterized the function of AtNHX5 and AtNHX6 in K⁺ and H⁺ homeostasis in Arabidopsis. Using a yeast expression system, we found that AtNHX5 and AtNHX6 recovered tolerance to high K⁺ or salt. We further found that AtNHX5 and AtNHX6 functioned at high K⁺ at acidic pH while AtCHXs at low K⁺ under alkaline conditions. In addition, we showed that the nhx5 nhx6 double mutant contained less K⁺ and was sensitive to low K⁺ treatment. Overexpression of AtNHX5 or AtNHX6 gene in nhx5 nhx6 recovered root growth to the wild-type level. Three conserved acidic residues, D164, E188, and D193 in AtNHX5 and D165, E189, and D194 in AtNHX6, were essential for K⁺ homeostasis and plant growth. nhx5 nhx6 had a reduced vacuolar and cellular pH as measured with the fluorescent pH indicator BCECF or semimicroelectrode. We further show that AtNHX5 and AtNHX6 are localized to Golgi and TGN. Taken together, AtNHX5 and AtNHX6 play an important role in K⁺ and pH homeostasis in Arabidopsis. Three conserved acidic residues are essential for K⁺ transport.     

Properties of pyruvate kinase from soybean nodule cytosol

The properties of pyruvate kinase from soybean (Glycine max L.) nodule cytosol were examined to determine what influence the N₂ fixation process might have on this supposed key control enzyme. A crude enzyme preparation was prepared by chromatography of cytosol extract on a diethylaminoethyl-cellulose column. ATP and citrate at 5 mm concentrations inhibited pyruvate kinase 27 and 34%, respectively. Enzyme activation was hyperbolic with respect to both K⁺ and NH₄⁺ concentrations. In the presence of physiological concentrations of K⁺ and high phosphoenolpyruvate (PEP) concentrations, NH₄⁺ inhibited enzyme activity. Comparisons of kinetic parameters (V_max and apparent K_a) for NH₄⁺ and K⁺ with inhibition curves indicated that inhibition was very likely a result of competition of the ions for activation site(s) on the pyruvate kinase. In addition, apparent K_a (monovalent cation) and K_m (PEP) were influenced by PEP and monovalent cation concentrations, respectively. This effect may reflect a fundamental difference between plant and animal pyruvate kinases. It is concluded that control of cytosol pyruvate kinase may be closely related to reactions involved in the assimilation of NH₄⁺.     

The α1 Na+−K+ pump of the Dahl salt-sensitive rat exhibits altered Na+ modulation of K+ transport in red blood cells

The properties of the α1 Na⁺-K⁺ pump were compared in Dahl salt-sensitive (DS) and salt-resistant (DR) strains by measuring ouabain-sensitive luxes (mmol/liter cell x hr = FU, Mean ± se) in red blood cells (RBCs) and varying internal ( _i ) and external ( _o ) Na⁺ and K⁺ concentrations. Kinetic parameters of several modes of operation, i.e., Na⁺/ K⁺, K⁺/K⁺, Na⁺/Na⁺ exchanges, were characterized and analyzed for curve-fitting using the Enzfitter computer program. In unidirectional flux studies (n=12 rats of each strain) into fresh cells incubated in 140 mm Na⁺ + 5 mm K⁺, ouabain-sensitive K⁺ influx was substantially lower in the DS than in DR RBCs, while ouabain-sensitive Na⁺ efflux and Na _i were similar in both strains. Thus, the coupling ratio between unidirectional Na⁺∶K⁺ fluxes was significantly higher in DS than in DR cells at similar RBC Na⁺ content. In the presence of 140 mm Na _o , activation of ouabain-sensitive K⁺ influx by K _o had a lower K _m and V _max in DS as estimated by the Garay equation (N=2.70 ± 0.33, K _m 0.74 ± 0.09 mm; V _max 2.87 ± 0.09 FU) than in DR rats (N=1.23 ± 0.36, K _m 2.31 ± 0.16 mm; v _max 5.70 ± 0.52 FU). However, the two kinetic parameters were similar following Na _o removal. The activation of ouabain-sensitive K⁺ influx by Na _i had significantly lower V _max in DS (9.3 ± 0.4 FU) than in DR (14.5 ± 0.6 FU) RBCs but similar K _m. These data suggest that the low K⁺ influx in DS cells is caused by a defect in modulation by Na _o and Na _i . Na⁺ efflux showed no differences in Na _i activation or trans effects by Na _o and K _o , thus accounting for the different Na⁺∶K⁺ coupling ratio in the Dahl strains. Further evidence for the differences in the coupling of ouabain-sensitive fluxes was found in studies of net Na⁺ and K⁺ fluxes, where the net ouabain-sensitive Na⁺ losses showed similar magnitudes in the two Dahl strains while the net ouabainsensitive K⁺ gains were significantly greater in the DR than the DS RBCs. Ouabain-sensitive Na⁺ influx and K⁺ efflux were also measured in these rat RBCs. The inhibition of ouabain-sensitive Na⁺ influx by K _o was fully competitive for the DS but not for the DR pumps. Thus, for DR pumps, K _o could activate higher K⁺ influx in DR pumps without a complete inhibition of ouabain-sensitive Na⁺ influx. This behavior is consistent with K _o interaction with distinct Na⁺ and K⁺ transport sites. In addition, the inhibition of K⁺ efflux by Na, was different between Dahl strains. Ouabain-sensitive K⁺ efflux at Na _i level of 4.6 mmol/liter cell, was significantly higher in DS (3.86 ± 0.67 FU) than in DR (0.86 ± 0.14 FU) due to a threefold higher K₅₀ for Na _i -inhibition 9.66 ± 0.41 vs. 3.09 ± 0.11 mmol/liter cell. This finding indicates that Na⁺ modulation of K⁺ transport is altered at both sides of the membrane. The dissociation of Na⁺ modulatory sites of K⁺ transport from Na⁺ transport sites observed in RBCs of Dahl strains suggests that K⁺ transport by the Na⁺-K⁺ pump is controlled by Na⁺ allosteric sites different from the Na⁺ transport sites. The alterations in K⁺ transport may be related to the amino acid substitution (Leu/Gln₂₇₆) reported for the cDNA of the α1 subunit of the Na⁺-K⁺ pump in the DS strain or to post-translational modifications during RBC maturation. These studies were supported by the following grants: NIH (HL-35664, HL-42120, HL-18318, HL-39267, HL-01967). J.R.R. is a Ford Foundation Predoctoral Fellow. A preliminary report of this work was presented at the International Conference on the Na⁺-K⁺ pump and 44th Annual Meeting of the Society of General Physiologists held at Woods Hole, MA, September 5–9, 1990, and published as an abstract in the J. Gen. Physiol. 96:70a, 1990.     

Specific modification of gastric K+-stimulated ATPase activity by thimerosal

Treatment of hog gastric microsomes with the sulfhydryl reagent, thimerosal (ethylmercurithiosalicylate), produced differential effects on the K⁺-ATPase and the K⁺-stimulated p-nitrophenylphosphatase activities. For example, exposure to 2 mM thimerosal for 3 min severely reduced the activity of K⁺-stimulated ATPase, while K⁺-p-nitrophenylphosphatase activity was enhanced 2- to 3-fold. Higher concentration of thimerosal, or longer incubation times, also led to inhibition of K⁺-p-nitrophenylphosphatase. The activated state of p-nitrophenylphosphatase could be sustained by a 20-fold, or greater, dilution of treated membranes, and could be reversed by reduction of membrane SH groups by exogenous thiols. Significant activation of K⁺-p-nitrophenylphosphatase was not produced by p-chloromercuribenzene sulfonate, p-chloromercuribenzoate or mersalyl; however, ethyl mercuric chloride had qualitatively similar activity effects as thimerosal. Kinetics of K⁺-p-nitrophenylphosphatase for thimerosal-treated membranes were altered as follows: V increased; K_m for p-nitrophenylphosphate unchanged for K_a for K⁺ increased. ATP, which is a potent inhibitor of K⁺-p-nitrophenylphosphatase activity in native membranes (K_I ≈ 200 μM). These data suggest that there are multiple SH groups which differentially influence the gastric K⁺-stimulated ATPase activity. Defined treatments with thimerosal are interpreted as an uncoupling of the K⁺-stimulated phosphatase component of the enzyme (for which p-nitrophenylphosphatase is a presumed model reaction). Such differential modifications can be usefully applied to the study of partial reactions of the enzyme and their specific role in the related H⁺-transport reaction.