Biophysical responses of red cell-membrane systems to very low concentrations of inorganic mercury

Changes in red blood cellsize, deformability, andosmotic fragility are indicators of altered condition and/or altered regulatory processes at the whole cell and membrane levels. An agent, such as HgCl₂, that brings about specific changes of this kind can therefore serve as a selective probe of such cell condition and regulatory state. Conversely, for a health-threatening agent “active” in this way, the cell-membrane responses serve to clarify the more fundamental bases of its toxicity, as well as to permit identification and characterization of its early and low-level actions on living systems. Taking advantage of recent advances in the technique of “resistive pulse spectroscopy,” we present a coordinated study of these three interrelated biophysical properties for the interactions of HgCl₂ with human red cells. We thereby are able to extend previous studies of this kind into domains of shorter time (instantaneous exposures), lower level exposures (down to 10⁻⁹ M, well below the level of acute human toxicity), as well as to additional kinds of responses (e.g., “dynamic osmotic hemolysis”). For conditions ranging from 10⁻⁴ to 10⁻⁹ M in HgCl₂, for instantaneous to 90-min-incubated exposures, for medium osmolarities from 120 to 300, the matrix of observed cell responses includes relative swelling as well as shrinkage, changes in deformability, and both enhancement of and protection against osmotic hemolysis. Some unexpected short-term effects of time and temperature of storage of blood cell stock samples, with respect to increasing and decreasing osmotic fragility, are also reported. These apparently disparate results are interpreted in terms of mercury interactions with cell and membrane SH groups, and a reasonable rationale is presented for most of the responses in terms of disruption of passive and active Na⁺−K⁺, gradient controls, plus interactions with cellular proteins.     

Effects of monovalent cations on phosphate accumulation and storage of the ectomycorrhizal fungus Suillus bovinus

Comparative in vivo ³¹P-NMR studies of the fungus Suillus bovinus (L.: Fr.) O. Kuntze in pure culture have produced interesting new data. To investigate the response of phosphate metabolism to a change in external monovalent cations, samples were exposed to a Hoagland solution containing different monovalent cations Li⁺, Na⁺, K⁺, or Rb⁺ at 10 mM concentration. A method of nutrient cycling during analysis where the cation was changed and the phosphate kept constant allowed us to determine the kinetics of phosphate accumulation, storage and incorporation into polyphosphate following exposure to the range of test cations. Different external monovalent cations had different effects upon changes in the content of both phosphate and polyphosphate. Treatment with Li⁺, Na⁺, or Rb⁺ resulted in a change in phosphate accumulation to 60, 73, and 107% and in content of the intracellular mobile polyphosphate (polyP) to 119, 112, and 94%, respectively, compared with the control taken as 100%. The effect of each cation is related to its position in the periodic table. Reversing this process, i.e., exchanging with K⁺, returned phosphate metabolism to normal. Although, the increase in depolarization of the cell membrane should affect the internal pH, fungal metabolism using energy requiring mechanisms appeared necessary to maintain the intracellular pH. Thus, increasing contents of mobile polyP were the consequence of an increasing energy demand. On the other hand, the increasing depolarization of the cell membrane following the sequence Rb⁺ < K⁺ < Na⁺ < Li⁺ inhibited the net P_i accumulation. Furthermore, it is postulated that the P_i accumulation was also regulated by the intracellular content in polyP.     

Thiol-Dependent passive K/Cl transport in sheep red cells: IV. Furosemide inhibition as a function of external Rb+, Na+, and Cl−

Summary The effect of the loop diuretic furosemide (4-chloro-N-furfuryl-5-sulfamoyl-anthranilic acid) on the thiol-dependent, ouabain-insensitive K(Rb)/Cl transport in low K⁺ sheep red cells was studied at various concentrations of extracellular Rb⁺, Na⁺ and Cl^–. In Rb⁺-free NaCl media, 2×10^–3 m furosemide inhibited only one-half of thiol-dependent K⁺ efflux. In the presence of 23mm RbCl, however, the concentration of furosemide to produce 50% K⁺ efflux inhibition (IC₅₀) was 5×10^–5 m. In Rb⁺ containing NaCl media, the inhibitory effect of 10^–3 m furosemide was equal to that caused by NO ₃ ^– replacement of Cl^– in the medium. The apparent synergistic action of furosemide and external Rb⁺ on K⁺ efflux was also seen in the ouabain-insensitive Rb⁺ influx. A preliminary kinetic analysis suggests that furosemide binding alters both maximal K⁺(Rb⁺) transport and apparent external Rb⁺ affinity. In the presence of external Rb⁺, Na⁺ (as compared to choline) exerted a small but significant augmentation of the furosemide inhibition of K⁺(Rb⁺) fluxes. There was no effect of Cl^– on the IC₅₀ value of furosemide. As there is no evidence for coupled Na⁺K⁺ cotransport in low K⁺ sheep red cells, furosemide may modify thiol-dependent K⁺(Rb⁺/Cl flux or Rb⁺ (and to a slight degree Na⁺) modulate the effect of furosemide.     

Measurement and stoichiometry of bumetanide-sensitive (2Na∶1K∶3Cl) cotransport in ferret red cells

Summary The bumetanide-sensitive uptake of Na⁺, K^–(Rb^–) and Cl^– has been measured at 21°C in ferrent red cells treated with (SITS+DIDS) to minimize anion flux via capnophorin (Band 3). During the time course of the influx experiments tracer uptake was a first-order rate process. At normal levels of external Na⁺ (150mm) the bumetanide-sensitive uptake of K⁺ was dependent on Cl^– and represented almost all of the K⁺ uptake, the residual flux demonstrating linear concentration dependence. The uptake of Na⁺ and Cl^– was only partially inhibited by bumetanide indicating that pathways other than (Na+K+Cl) cotransport participate in these fluxes. The diuretic-sensitive uptake of Na⁺ or Cl^– was, however, abolished by the removal of K⁺ or the complementary ion indicating that bumetanide-sensitive fluxes of Na⁺, K⁺ and Cl^– are closely coupled. At very low levels of [Na] _o (<5mm) K⁺ influx demonstrated complex kinetics, and there was evidence of the unmasking of a bumetanide-sensitive Na⁺-independent K⁺ transport pathway. The stoichiometry of bumetanide-sensitive tracer uptake was 2Na1K3Cl both in cells suspended in a low and a high K⁺-containing medium. The bumetanide-sensitive flux was markedly reduced by ATP depletion. We conclude that a bumetanide-sensitive cotransport of (2Na1K3Cl) occurs as an electroneutral complex across the ferret red cell membrane.     

Effects of monovalent cations on the activities associated with coupling site III of rat liver mitochondria

The effects of substitution of K⁺ by Li⁺, Na⁺, or Rb⁺ in the assay medium on the processes of electron transfer and H⁺ translocation associated with Site III are investigated. The replacement of K⁺ with Rb⁺ has little effect, if any, on the measured initial rates of H⁺ extrusion and electron transfer. The substitution of K⁺ by Li⁺ increases the initial rate of both processes simultaneously while the replacement of K⁺ by Na⁺ causes an enhancement on the rate of electron transfer with concomitant inhibition of the observed acidification. The presence of either Na⁺ or Li⁺ decreases the proton-leak rate of the inner membrane. These results suggest that the link between electron transfer and H⁺ translocation in Site III is weakened by the presence of Na⁺.     

Generation of plasma membrane potential by the Na+-pump coupled to proton extrusion

Lettré cells maintain a plasma membrane potential near — 60mV, yet are scarcely depolarized by 80 mM Rb⁺ and are relatively impermeable to ⁸⁶Rb⁺. They are depolarized by ouabain without a concomitant change in intracellular cation content. Addition of K⁺ to cells suspended in a K⁺ free medium, or of Na⁺ to cells in a Na⁺ free medium, hyperpolarizes the cells. They contain electroneutral transport mechanisms for Na⁺, K⁺ and H⁺ which can function as Na⁺:K⁺ and Na⁺:H⁺ exchanges. It is concluded that plasma membrane potential of Lettré cells, in steady-state for Na⁺ and K⁺, is produced by an electrogenic Na⁺ pump sustained by electroneutral exchanges, and restricted by anion leakage.     

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

Whereas cation transport by the electrogenic membrane transporter Na⁺,K⁺-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H⁺,K⁺-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb⁺ or Li⁺ transport by Na⁺,K⁺- or gastric H⁺,K⁺-ATPase in single cells. Using Xenopus oocytes as expression system, we determine the amount of Rb⁺ (Li⁺) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb⁺ uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g. to quantitatively determine the 3Na⁺/2K⁺ transport stoichiometry of the Na⁺,K⁺-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H⁺,K⁺-ATPase. In principle, the assay is not limited to K⁺-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.     

The mechanism of cation permeation in rabbit gallbladder

Summary The questions underlying ion permeation mechanisms, the types of experiments available to answer these questions, and the properties of some likely permeation models are examined, as background to experiments designed to characterize the mechanism of alkali cation permeation across rabbit gallbladder epithelium. Conductance is found to increase linearly with bathing-solution salt concentrations up to at least 400mm. In symmetrical solutions of single alkali chloride salts, the conductance sequence is K⁺>Rb⁺>Na⁺>Cs⁺∼Li⁺. The current-voltage relation is linear in symmetrical solutions and in the presence of a single-salt concentration gradient up to at least 800 mV. The anion/cation permeability ratio shows little change with concentration up to at least 300mm. Ca⁺⁺ reduces alkali chloride single-salt dilution potentials, the magnitude of the effect being interpreted as an inverse measure of cation equilibrium constants. The equilibrium-constant sequence deduced on this basis is K⁺>Rb⁺>Na⁺∼Cs⁺∼Li⁺. These results suggest (1) that the mechanism of cation permeation in the gallbladder is not the same as that in a macroscopic ion-exchange membrane; (2) that cation mobility ratios are closer to one than are equilibrium-constant ratios; (3) that the rate-limiting step for cation permeation is in the membrane interior rather than at the membrane-solution interface; and (4) that the rate-controlling membrane is one which is sufficiently thick that it obeys microscopic electroneutrality.     

Sodium and potassium interactions with Na+-ATPase of inside-out membrane vesicles from high-K+ and low-K+ sheep red cells

Na⁺-ATPase of high-K⁺ and low-K⁺ sheep red cells was examined with respect to the sidedness of Na⁺ and K⁺ effects, using inside-out membrane vesicles and very low ATP concentrations (?2 μM). With varying amounts of Na⁺ in the medium, i.e., at the cytoplasmic surface, Na_cyt⁺, the activation curves show that high-K⁺ Na⁺-ATPase has a higher affinity for Na_cyt⁺ compared to low-K⁺. The apparent affinity for Na_cyt⁺ is also increased by increasing the ATP concentrations in high-K⁺ but not low-K⁺. With Na_cyt⁺ present, Na⁺-ATPase is stimulated by intravesicular Na⁺, i.e., Na⁺ at the originally external surface, Na_ext⁺, to a greater extent in low-K⁺ than high-K⁺. Intravesicular K⁺ (K_ext⁺) activates Na⁺-ATPase in high-K⁺ but not in low-K⁺ vesicles and extravesicular K⁺ (K_cyt⁺) inhibits low-K⁺ but not high-K⁺ Na⁺-ATPase. Thus, the genetic difference between high-K⁺ and low-K⁺ is expressed as differences in apparent affinities for both Na⁺ and K⁺ and these differences are evident at both cytoplasmic and external membrane surfaces.     

Potassium requirements ofSaccharomyces cerevisiae

The growth rate ofSaccharomyces cerevisiae was dependent on K⁺ content in culture medium in a certain range of K⁺ concentrations. Above the upper limit of the range, growth did not respond to K⁺ increase, and below the lower limit, yeast died. Rb⁺ and Na⁺ enhanced growth in the range of K⁺ dependence and decreased the K⁺ concentration below which cells died. Both Rb⁺ and Na⁺ became toxic above a certain Rb⁺/K⁺ and Na⁺/K⁺ cellular ratio.     

Nigericin forms highly stable complexes with lithium and cesium

Nigericin is a monocarboxylic polyether molecule described as a mobile K⁺ ionophore unable to transport Li⁺ and Cs⁺ across natural or artificial membranes. This paper shows that the ion carrier molecule forms complexes of equivalent energy demands with Li⁺, Cs⁺, Na⁺, Rb⁺, and K⁺. This is in accordance with the similar values of the complex stability constants obtained from nigericin with the five alkali metal cations assayed. On the other hand, nigericinalkali metal cation binding isotherms show faster rates for Li⁺ and Cs⁺ than for Na⁺, K⁺, and Rb⁺, in conditions where the carboxylic proton does not dissociate. Furthermore, proton NMR spectra of nigericin-Li⁺ and nigericin-Cs⁺ complexes show wide broadenings, suggesting strong cation interaction with the ionophore; in contrast, the complexes with Na⁺, K⁺, and Rb⁺ show only clear-cut chemical shifts. These latter results support the view that nigericin forms highly stable complexes with Li⁺ and Cs⁺ and contribute to the explanation for the inability of this ionophore to transport the former cations in conditions where it catalyzes a fast transport of K⁺>Rb⁺>Na⁺.Part of the results of this paper were presented at the 14th International Congress of Biochemistry in Prague, Czechoslovakia.     

Sulfatide role in the sodium pump

Summary Sodium efflux was studied in²²Na-loaded red blood cells in the presence of arylsulfatase, an enzyme that specifically hydrolyzes sulfatide. Sodium efflux was inhibited in proportion to the amount of arylsulfatase present. Maximum inhibition was almost as high as the efflux obtained in medium with K⁺ absent. At maximum inhibition 83.2% of the sulfatide content of the fragmented red blood cell membranes was hydrolyzed and ouabain-sensitive (Na⁺+K⁺)-ATPase activity was inhibited by 100%. Sodium efflux, sulfatide content, and (Na⁺+K⁺)-ATPase activity were unaffected with arylsulfatase in the presence of a high concentration of sulfatide. These results indicate that sulfatide plays a specific role in sodium and potassium ion transport. They also suggest that most sulfatide is localized externally in the red blood cell membrane.     

Kinetic parameters and mechanism of active cation transport in HeLa cells as studied by Rb+ influx

On incubation of HeLa cells in chilled isotonic medium, intracellular Na⁺ (Na_c⁺) increased and K⁺ (K_c⁺) decreased with time, reaching steady levels after 3 h. The steady levels varied in parallel with the extracellular cation concentrations ([Na⁺]_e, [K⁺]_e). The cell volumes and the protein and water contents, respectively, of cells kept for 3 h in chilled media of various [Na⁺]_e and [K⁺]_e were not significantly different. Ouabain-sensitive Rb⁺ influx took place at the initial rate for a certain period which depended on [Na⁺]_c at the beginning of the assays. The existence of two external K⁺ loading sites per Na⁺/K⁺-pump was demonstrated. The affinities of the sites for Rb⁺ as a congener of K⁺ were almost the same. Na_e⁺ inhibited ouabain-sensitive Rb⁺ influx competitively, whereas K_c⁺ was not inhibitory. Kinetic parameters were determined: the $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for Rb_e⁺ in the absence of Na_e⁺ was 0.16 mM and the K_i for Na_e⁺ was 36.8 mM; the $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ was 19.5 mM and the K_i for K_c⁺ seemed to be extremely large. The rate equation of the ouabain-sensitive Rb⁺ influx suggests that Na⁺ and K⁺ are exchanged alternately through the pump by a binary mechanism.     

Selectivity and permeation of alkali metal ions in K+-channels

Ion conduction in K⁺-channels is usually described in terms of concerted movements of K⁺ progressing in a single file through a narrow pore. Permeation is driven by an incoming ion knocking on those ions already inside the protein. A fine-tuned balance between high-affinity binding and electrostatic repulsive forces between permeant ions is needed to achieve efficient conduction. While K⁺-channels are known to be highly selective for K⁺ over Na⁺, some K⁺ channels conduct Na⁺ in the absence of K⁺. Other ions are known to permeate K⁺-channels with a more moderate preference and unusual conduction features. We describe an extensive computational study on ion conduction in K⁺-channels rendering free energy profiles for the translocation of three different alkali ions and some of their mixtures. The free energy maps for Rb⁺ translocation show at atomic level why experimental Rb⁺ conductance is slightly lower than that of K⁺. In contrast to K⁺ or Rb⁺, external Na⁺ block K⁺ currents, and the sites where Na⁺ transport is hindered are characterized. Translocation of K⁺/Na⁺ mixtures is energetically unfavorable owing to the absence of equally spaced ion-binding sites for Na⁺, excluding Na⁺ from a channel already loaded with K⁺.     

Potassium and Sodium Fluxes in the PhytopathogenicFungus Fusarium oxysporum var. lini

Rubidium uptake in potassium-starved cells followed biphasic kinetics in the micromolar and millimolar range and was independent of the temperature. In contrast, Rb⁺ uptake in normal-K⁺ cells followed a monophasic kinetics in the millimolar range and increased at temperatures higher than 30°C. Differences in the K _m values and in the Arrhenius plots of Rb⁺ uptake suggest different uptake systems in K⁺-starved and in normal-K⁺ cells. In addition, the substantial inhibition of Rb⁺ uptake caused by carbonyl cyanide-m-chlorophenyl hydrazone indicates that these systems are strongly dependent on membrane voltage. Lithium (sodium) tolerance, influx, and efflux were separately studied. F. oxysporum was shown to be very tolerant to sodium, while lithium caused a specific toxic effect. Li⁺ uptake in K⁺-starved cells exhibits a monophasic kinetics with low affinity. Li⁺ efflux was not affected by external pH or addition of potassium to the medium, suggesting that a Na⁺/cation antiporter is not involved in this process. Received: 14 March 2000 / Accepted: 5 June 2000     

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.     

Requirement of monovalent cations in the acrosome reaction of guinea pig spermatozoa

The majority of the spermatozoa precapacitated in Ca²⁺-free medium underwent the acrosome raction rapidly when they were transferred to Ca²⁺-containing medium. The presence of Na⁺ and Ca²⁺ in the medium was essential for the acrosome reaction. The vast majority of spermatozoa failed to undergo the reaction in Ca²⁺ medium lacking monovalent ions, although they remained motile. At the concentration of 140 mM, Na⁺, K⁺, Rb⁺, and Cs⁺ all supported the reaction at the maximum level, but at 50 mM the latter three ions were not as effective as Na⁺. Li⁺ was least effective in supporting the reaction. Virtually no acrosome reactions took place when precapacitated spermatozoa were first exposed to Na⁺ medium (no Ca²⁺) and then to Ca²⁺ medium (no Na⁺). On the other hand, a considerably higher proportion of spermatozoa acrosome reacted when they were exposed to these media in the reverse order. The most efficient acrosome reactions took place when the medium contained both a monovalent ion (Na⁺) and Ca²⁺ simultaneously. Possible mechanisms by which monovalent and divalent cations participate in the acrosome reaction are discussed.     

Inhibition of Na+/K+-ATPase may be one mechanism contributing to potassium efflux and cell shrinkage in CD95-induced apoptosis

To investigate the involvement of K⁺ efflux in apoptotic cell shrinkage, we monitored efflux of the K⁺ congener,⁸⁶ Rb⁺, and cell volume during CD95-mediated apoptosis in Jurkat cells. An anti-CD95 antibody caused apoptosis associated with intracellular GSH depletion, a significant increase in ⁸⁶Rb⁺ efflux, and a decrease in cell volume compared with control cells. Preincubating Jurkat cells with Val-Ala-Asp-chloromethylketone (VAD-cmk), an inhibitor of caspase proteases, prevented the observed ⁸⁶Rb⁺ efflux and cell shrinkage induced by the anti- CD95 antibody. A wide range of inhibitors against most types of K⁺ channels could not inhibit CD95-mediated efflux of⁸⁶ Rb⁺, however, the uptake of⁸⁶ Rb⁺ by Jurkat cells was severely compromised when treated with anti-CD95 antibody. Uptake of⁸⁶ Rb⁺ in Jurkat cells was sensitive to ouabain (a specific Na⁺/K⁺-ATPase inhibitor), demonstrating Na⁺/K⁺-ATPase dependent K⁺ uptake. Ouabain induced significant⁸⁶ Rb⁺ efflux in untreated cells, as well as it seemed to compete with⁸⁶ Rb⁺ efflux induced by the anti-CD95 antibody, supporting a role for Na⁺/K⁺-ATPase in the CD95-mediated⁸⁶ Rb⁺ efflux. Ouabain treatment of Jurkat cells did not cause a reduction in cell volume, although together with the anti-CD95 antibody, ouabain potentiated CD95-mediated cell shrinkage. This suggests that the observed inhibition of Na⁺+/K⁺-ATPase during apoptosis may also facilitate apoptotic cell shrinkage.     

Ba2+-inhibitable86Rb+ fluxes across membranes of vesicles from toad urinary bladder

Summary ⁸⁶Rb⁺ fluxes have been measured in suspensions of vesicles prepared from the epithelium of toad urinary bladder. A readily measurable barium-sensitive, ouabain-insensitive component has been identified; the concentration of external Ba²⁺ required for half-maximal inhibition was 0.6mm. The effects of externally added cations on⁸⁶Rb⁺ influx and efflux have established that this pathway is conductive, with a selectivity for K⁺, Rb⁺ and Cs⁺ over Na⁺ and Li⁺. the Rb⁺ uptake is inversely dependent on external pH, but not significantly affected by internal Ca²⁺ or external amiloride, quinine, quinidine or lidocaine. It is likely, albeit not yet certain, that the conductive Rb⁺ pathway is incorporated in basolateral vesicles oriented right-side-out. It is also not yet clear whether this pathway comprises the principle basolateral K⁺ channel in vivo, and that its properties have been unchanged during the preparative procedures. Subject to these caveats, the data suggest that the inhibition by quinidine of Na⁺ transport across toad bladder does not arise primarily from membrane depolarization produced by a direct blockage of the basolateral channels. It now seems more likely that the quinidine-induced elevation of intracellular Ca²⁺ activity directly blocks apical Na⁺ entry.     

Author index

The stimulation of ouabain-sensitive Na⁺ efflux by external Na⁺, K⁺ and Li⁺ was studied in control and ATP-depleted human red cells. In the presence of 5 mM Na⁺, with control and depleted cells, Li⁺ stimulated with a lower apparent affinity than K⁺, and gave a smaller maximal activation than K⁺. The ability of Na⁺, K⁺ and Li⁺ to activate Na⁺ efflux was a function of the ATP content of the cells. Relative to K⁺ both Na⁺ and Li⁺ became more effective activators when the ATP was reduced to about one tenth of the control values. At this low ATP concentration Na⁺ was absolutely more effective than K⁺.