Characterization of Na+-coupled glutamate/aspartate transport by a rat brain astrocyte line expressing GLAST and EAAC1.

D-aspartate (D-Asp) uptake by suspensions of cerebral rat brain astrocytes (RBA) maintained in long-term culture was studied as a means of characterizing function and regulation of Glutamate/Aspartate (Glu/Asp) transporter isoforms in the cells. A-asp influx is Na+-dependent with Km = 5 microm and Vmax = 0.7 nmoles x min(-1) x mg protein-1. Influx is sigmoidal as f[Na+] with Na+Km approximately 12 microm and Hill coefficient of 1.9. The cells establish steady-state D-Asp gradients >3,000-fold. Phorbol ester (PMA) enhances uptake, and gradients near 6,000-fold are achieved due to a 2-fold increase in Vmax, with no change in Km. At initial [D-Asp] = 10 microm, RBA take up more than 90% of total D-Asp, and extracellular levels are reduced to levels below 1 microm. Ionophores that dissipate the Delta(mu)Na+ inhibit gradient formation. Genistein (GEN, 100 microm), a PTK inhibitor, causes a 40% decrease in d-Asp. Inactive analogs of PMA (4alpha-PMA) and GEN (daidzein) have no detectable effect, although the stimulatory PMA response still occurs when GEN is present. Further specificity of action is indicated by the fact that PMA has no effect on Na+-coupled ALA uptake, but GEN is stimulatory. d-Asp uptake is strongly inhibited by serine-O-sulfate (S-O-S), threohydroxy-aspartate (THA), L-Asp, and L-Glu, but not by D-Glu, kainic acid (KA), or dihydrokainate (DHK), an inhibition pattern characteristic of GLAST and EAAC1 transporter isoforms. mRNA for both isoforms was detected by RT-PCR, and Western blotting with appropriate antibodies shows that both proteins are expressed in these cells.

Na+-coupled alanine transport in LLC-PK1 cells: the relationship between the Km for Na+ at low [Alanine] and potential dependence for the system.

Analysis of the mechanistic basis by which sodium-coupled transport systems respond to changes in membrane potential is inherently complex. Algebraic expressions for the primary kinetic parameters (Km and Vmax) consist of multiple terms that encompass most rate constants in the transport cycle. Even for a relatively simple cotransport system such as the Na+/alanine cotransporter in LLC-PK1 cells (1:1 Na+ to substrate coupling, and an ordered binding sequence), the algebraic expressions for Km for either substrate includes ten of the twelve rate constants necessary for modeling the full transport cycle. We show here that the expression of Km of the first-bound substrate (Na+) simplifies markedly if the second-bound substrate (alanine) is held at a low concentration so that its' binding becomes the rate limiting step. Under these conditions, the expression for the KNam includes rate constants for only two steps in the full cycle: (i) binding/dissociation of Na+, and (ii) conformational 'translocation' of the substrate-free protein. The influence of imposed changes in membrane potential on the apparent KNam for the LLC-PK1 alanine cotransporter at low alanine thus provides insight to potential dependence at these sites. The data show no potential dependence for KNam at 5 micron alanine, despite marked potential dependence at 2 mm alanine when the full algebraic expression applies. The results suggest that neither translocation of the substrate-free form of the transporter nor binding/dissociation of extracellular sodium are potential dependent events for this transport system.

The molecular mechanism and potential dependence of the Na+/glucose cotransporter.

Activity of the Na+/glucose cotransporter endogenously expressed in LLC-PK1 cells was measured using whole cell recording techniques under three different sodium concentration conditions: 1) externally saturating, zero trans; 2) 40 mM external, zero trans; and 3) externally saturating, 50 mM trans. Activity of the transporter with increasing concentrations of sugar was measured for each set of conditions, from which the maximal current for saturating sugar, Im, was determined. The Im measured shows substantial potential dependence for each set of conditions. The absolute Im and the relative potential dependence of Im compared among the various solute conditions were used to identify which loci in the transport cycle are responsible for potential-dependent changes in function. The experimental data were compared with the predicted Im values calculated from an eight-state, sequential, reversible model of a transport reaction kinetic scheme. Predictions derived from assignment of rate limitation and/or potential dependence to each of the 16 transitions in the transport pathway were derived and compared with the measured data. Most putative models were dismissed because of lack of agreement with the measured data, indicating that several steps along the transport pathway are not rate limiting and/or not potential dependent. Only two models were found that can completely account for the measured data. In one case, translocation of the free carrier must be rate limiting, and both extracellular sodium-binding events as well as translocation of both free and fully loaded carrier forms must be potential-dependent transitions. In the second case, translocation of the free carrier and dissociation of the first sodium to be released intracellularly must be equivalently rate limiting. In this case only the two translocation events are required to be potential dependent. The two external sodium-binding events might still be potential dependent, but this is not required to fit the data. Previous reports suggest that the first model is correct; however, no direct experimental data compel us to dismiss the second option as a feasible model.

A model for the kinetic mechanism of sodium-coupled L-alanine transport in LLC-PK1 cells.

The kinetics of sodium-dependent L-alanine transport were characterized in ATP-depleted LLC-PK1 cells, which allows experimental imposition of an interior negative diffusion potential across the plasma membrane. Under these conditions a wide range of sodium concentrations can be studied without altering the membrane potential. When Na+ is the variable substrate, the apparent maximal velocity (V max) for transport changes nearly fourfold for the five different alanine concentrations studied (0.05-2.0 mM). In contrast, at five different sodium concentrations, ranging from 10 to 135 mM, the apparent V max with variable alanine remains nearly constant at 5.3 +/- 1.2 nmol.min-1.mg cell protein-1. The ratio of the two primary kinetic parameters, Michaelis constant (Km)/V max, varies markedly no matter which solute is treated as the variable illustrate. These data are consistent with a simultaneous ordered transport mechanism in which sodium binds before alanine to the transport protein at the extracellular surface of the membrane. Alanine-dependent 22Na+ influx is more than five times faster if unlabeled intracellular sodium is present than in its absence. Sodium-dependent influx of [14C]alanine is more rapid than net alanine flux only if unlabeled Na+ and alanine are both present intracellularly. These results indicate that the cotransporter can function more rapidly in an exchange mode than when it catalyzes net solute uptake and that Na+ is the first solute to be released at the intracellular side of the membrane. A model is presented that can be used for further quantitative analysis of the kinetic and functional properties of the cotransport system.

Na(+)-coupled alanine transport in LLC-PK1 cells.

Transport of alanine (Ala) was characterized in LLC-PK1 renal epithelial cells. Transport capability for Ala falls by 75% in postconfluent cultures, while Na(+)-coupled alpha-methylglucoside (AMG) transport rises more than fourfold during the same interval. The kinetics of Ala transport were characterized in ATP-depleted cells to allow experimental imposition of changes in Na+ gradient and control of membrane potential across the plasma membrane. At 100 microM Ala and 135 mM Na+, > 98% of the unidirectional Ala influx is dependent on the presence of Na+ in cells from postconfluent cultures. Li+ is only 1% as effective as Na+, and other monovalent cations are ineffective in supporting Ala uptake. alpha-(Methylamino)isobutyric acid (MeAIB; 5 mM) causes only a small inhibition (approximately 10%) of 100 microM Ala influx. The low selectivity for Li+; low sensitivity to competition by MeAIB or aminoisobutyric acid; pronounced inhibition by serine, homoserine, cysteine, homocysteine and threonine; moderate inhibition by valine, isoleucine, proline and histidine; and lack of inhibition by lysine, arginine, and aspartate are more consistent with those characteristics reported for entry via the ASC amino acid transport system rather than those associated with the A system. Alanine influx exhibits a hyperbolic relationship with increasing Ala or Na+ concentration. Kinetic analysis suggests a single transport pathway with a Michaelis constant (Km) for alanine of 380 microM (when Na+ is 135 mM), apparent Km for Na+ of 32 mM (with 100 microM Ala), and a maximum velocity of 7 nmol.min-1.mg cell protein-1. An interior-negative diffusion potential induces a similar enhancement of [14C]alanine or [14C]tetraphenylphosphonium influx (approximately 40%). In contrast, AMG influx is enhanced by a factor of 2.2 under the same conditions. AMG uptake also shows a sigmoidal relationship with Na+ concentration. Hill coefficients are 1.56 for AMG and 1.0 for alanine. Direct measurement of Na(+)-Ala coupling stoichiometry yields a value of 1.01 +/- 0.07. Under the same conditions, Na(+)-AMG coupling stoichiometry is 2.1 +/- 0.25. The difference in coupling stoichiometries provides an explanation for differences in intensity of interaction between Na(+)-coupled transport systems for sugars and amino acids.

Na+ binding to the Na(+)-glucose cotransporter is potential dependent.

Activity of the Na(+)-glucose cotransporter in LLC-PK1 epithelial cells was assayed by measuring sugar-induced currents (IAMG) using whole cell recording techniques. IAMG was compared among cells by standardizing the measured currents to cell size using cell capacitance measurements. IAMG at a given membrane potential was measured as a function of alpha-methylglucoside (AMG) concentration and can be fit to Michaelis-Menten kinetics. IAMG at varying Na+ concentrations can be described by the Hill equation with a Hill coefficient of 1.6 at all tested potentials. At high external Na+ levels (155 mM), Na+ is at least 90% saturating at all tested potentials. Maximal currents at a given membrane potential (Im) are calculated from the Michaelis-Menten equation fit to data measuring IAMG vs. AMG concentration at a constant Na+ concentration. Im showed potential dependence under all conditions. Potential-dependent Na+ binding rate(s) cannot alone explain the observed potential dependence of Im under saturating Na+ conditions. Therefore, because Im is potential dependent, at least one step of the transport cycle other than external Na+ binding must be potential dependent. Im was also calculated from data taken at 40 mM external Na+. At all potentials studied, Im at 155 mM Na+ is greater than Im calculated at 40 mM Na+. This implies that the rate of external Na+ binding to the transporter at 40 mM also affects the maximal transport rate. Furthermore, Im at 40 mM external Na+ increases with hyperpolarization faster than Im at 155 mM Na+. Together, these facts indicate that the rate at which Na+ binds to the transporter is also potential dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of ATP-sensitive K+ channels in pancreatic beta-cells by nonsulfonylurea drug linogliride.

Linogliride is a nonsulfonylurea drug that lowers blood glucose levels in nondiabetic and diabetic humans and animals. Linogliride also stimulates insulin release in vitro. In the perfused pancreas, pretreatment with tolbutamide desensitizes beta-cells to the action of linogliride. We tested the hypothesis that linogliride, like tolbutamide, affects the activity of ATP-sensitive K+ channels, which are thought to control insulin release. We used the whole-cell voltage-clamping technique to measure the K+ current through ATP-sensitive K+ channels in the plasma membrane of single rat beta-cells, which were dialyzed with 30 microM ATP. Linogliride (10-300 microM) inhibited the K+ current; half-maximal inhibition was observed at 6-25 microM, depending on how much time was allowed for equilibration of the drug. Reversal of the inhibition was slow (t1/2 approximately 4 min). In summary, linogliride leads to a decrease in the activity of ATP-sensitive K+ channels.

Sodium-dependent succinate transport by isolated chick intestinal cells.

Isolated chick intestinal epithelial cells take up succinate by a Na(+)-coupled transport system similar in some characteristics to those described for renal epithelium. The transport system exhibits a hyperbolic dependence on succinate concentration but a sigmoidal dependence on Na+ concentration. Best nonlinear fit of the Na+ dependence data to the Hill equation indicates a Michaelis constant for half-maximal transport rate (Km) for Na+ of approximately 20 mM, a maximal transport rate (Vmax) of 1.1 nmol succinate.min-1.mg protein-1, and a Hill coefficient of 2.5. Nearly equivalent fit was obtained with trial Hill coefficients down to 2.0. The data for succinate dependence indicated a Km of 25 microM and Vmax of 1.05 nmol.min-1.mg protein-1. The kinetic parameters indicate a higher affinity, lower capacity system than for succinate transport in the renal brush-border system. Thiocyanate-induced diffusion potentials cause no change in Na(+)-dependent succinate influx despite pronounced effects on the influx of tetraphenylphosphonium and on Na(+)-dependent alpha-methylglucoside (AMG) and alanine uptake. Several other dicarboxylic and tricarboxylic metabolic intermediates (but not the dicarboxylic amino acids) compete with succinate for uptake via the transport system. The data are consistent with the likelihood that these cells have a succinate transport system with a 2Na+:1succinate stoichiometry per transport cycle. The system catalyzes no net charge transfer and is therefore different from the potential-responsive succinate transporter described for renal tissue.

Whole cell recording of sugar-induced currents in LLC-PK1 cells.

Gigaohm-seal whole cell recording techniques were used to monitor function of the Na(+)-coupled sugar transport system in LLC-PK1 cells. The currents coupled to sugar transport were identified as those that are induced by the presence of 10 mM alpha-methylglucoside (AMG) in either the extracellular or intracellular compartment and were inhibited by addition of 320-800 microM phlorizin to the extracellular bathing medium. The sugar-induced currents are small, 15-20 pA, but of the expected magnitude as determined from the known kinetic parameters for Na(+)-coupled sugar transport in LLC-PK1 cells. The phlorizin-sensitive currents are Na+ dependent and can be studied under conditions in which the net Na+ and sugar flux (and consequently the Na+ electrical current) is in either the inward or outward direction. The reversal potential of the sugar-induced currents measured under conditions with high Na+ and AMG concentrations inside the cell is close to values predicted from thermodynamic principles, assuming a coupling stoichiometry of 2 Na+: 1 sugar for the transport system. The reversal potential of the sugar-induced currents with high extracellular Na+ and AMG is not equal to the predicted value, but it is of the polarity expected for inward-imposed solute gradients. Reasons for the observed discrepancy between observed and calculated values are discussed.

Isolation of intestinal epithelial cells and evaluation of transport functions.

Epithelial cells can be isolated from the small intestine of chickens by a procedure involving hyaluronidase treatment of the intact tissue. The isolated cells retain a high degree of functional activity as assessed by the formation of 70-fold gradients of alpha-MG. Stability of the sugar gradients reflects maintenance of stable electrochemical Na+ gradients across the plasma membrane. The cells can be used to evaluate the properties of Na(+)-dependent sugar transport, Na(+)-independent sugar transport, ion transport, metabolism, membrane potentials, and the integration of these events, all of which are important to achieving a stable sugar gradient.

Effect of saccharin on the ATP-induced increase in Na+ permeability in isolated chicken intestinal epithelial cells.

When isolated intestinal cells from 3-wk-old chickens are treated with exogenous ATP they undergo a dramatic increase in permeability towards Na+. The increase occurs instantaneously and maximum cell loading with Na+ occurs within 2 min. The response is dose dependent (0.1-1.0 mM-ATP) and results in as much as a 10-fold increase in unidirectional influx of 22Na+ into the cells. The resting cellular Na+ gradient and membrane potential are partially dissipated and consequently Na+-dependent transport of sugars and amino acids is inhibited. Sodium saccharin (20 mM), added at the same time as ATP, completely blocks the effect of ATP on Na+ permeability and preserves the functional capacity of the cells for Na+-dependent sugar or amino acid transport. Partial protection is afforded by 10 mM-saccharin. Saccharin added 2 min after ATP will reverse the enhanced Na+ permeability that has already been induced. In cells that have not been treated with ATP, saccharin induces enhanced sugar and amino acid gradients (P less than 0.05 in paired comparisons from the same cell preparation), indicating that it may also inhibit Na+ permeability of the unperturbed membrane and allow cells to establish higher Na+ gradients and/or membrane potentials. The effect of saccharin in blocking ATP-induced Na+ permeability occurs within 10 sec and at a much lower dose than that required for blockade of facilitated diffusional sugar transfer in these cells.

Low-affinity intestinal L-aspartate transport with 2:1 coupling stoichiometry for Na+/Asp.

Epithelial cells isolated from chick small intestine were used to define the ionic and electrical characteristics of a low-affinity (Km = 4.1 mM) L-aspartate transport system. L-Glutamate and D-aspartate, but not D-glutamate, were found to inhibit L-aspartate influx, suggesting that this uptake system has a substrate specificity similar to that previously described for a high-affinity (Km = 16 microM) acidic amino acid transporter in the same cells. Low-affinity uptake is Na+ dependent with a Hill coefficient (n) of 1.4. Intracellular K+ moderately enhances but is not required for aspartate influx, and this response is modulated by changes in intracellular pH. The Na+-dependent uptake of aspartate is electroneutral, as evidenced by insensitivity to pronounced changes in delta psi induced by anion gradients or valinomycin in the presence of K+ gradients. Because the above characteristics can be consistent with several transport models, direct measurement of delta Na+-delta Asp coupling stoichiometry were performed. The coupling ratio was determined to be approximately 2.0. A model for intestinal Na+-dependent L-Asp transport is suggested in which each transport cycle involves inward transfer of 2Na+:1Asp+ and outward transfer of K+ or H+ in a net electroneutral set of events.

Inhibition of the serosal sugar carrier in isolated intestinal epithelial cells by saccharin.

Isolated intestinal cells accumulate certain monosaccharides via an Na+-dependent, active transport system localized in the brush-border membrane, and release sugar molecules at the basolateral boundary via a facilitated diffusion, passive system. Work described here indicates that sodium saccharin (25-130 mM) has little if any direct effect on the active transport system, but that the passive transport system is inhibited by saccharin. A short period of exposure (10-60 min) is required for expression of the effect, which is detectable at saccharin concentrations as low as 10 mM. At 100 mM-sodium saccharin, as much as 50% inhibition occurs. Saccharin also appears to act as a weak metabolic inhibitor. The basis of the 'non-specific' effect is not understood, but it can compromise the capacity of the epithelial cells to form sugar gradients. When a sugar is accumulated that satisfies both transport systems (for example 3-O-methylglucose) the effect of saccharin on the passive transport system is the predominant one, and the cells establish a higher sugar gradient than that observed in the absence of saccharin. The 'non-specific' metabolic effect is manifested as an inhibition of sugar gradient formation when sugars that satisfy only the active system (such as alpha-methylglucoside) are accumulated.

Na+-coupled sugar transport: membrane potential-dependent Km and Ki for Na+.

Kinetic analysis of the characteristics of phlorizin binding and of the Na+, sugar, and potential dependence of alpha-methylglucoside (alpha-MG) influx into isolated avian intestinal cells has pointed toward two alternative models for the transport mechanism (D. Restrepo and G. A. Kimmich, J. Membr. Biol. 89: 269-280, 1986). One of these models envisions a potential-dependent Na+ binding event (Na+ well concept) as a part of the molecular mechanism. The data reported here show that the apparent Km for Na+ for sugar transport is sharply dependent on the magnitude of the membrane potential. When intracellular Na+ is absent, the maximal velocity (Vmax) achieved for sugar influx is the same with or without a potential, although Vmax is obtained at a lower Na+ concentration when a potential is imposed (interior negative). Intracellular Na+ severely inhibits the influx of sugar in the absence of a potential, but this effect is largely overcome when a potential is present. The Vmax obtained when intracellular Na+ is present is a function of the potential. These results are consistent with a transport model in which Na+ binding to the Na+-dependent sugar carrier at the extracellular surface of the membrane and debinding at the inner surface of the membrane are both potential-dependent events.

SITS-sensitive Cl- conductance pathway in chick intestinal cells.

The unidirectional influx of 36Cl- into isolated chick epithelial cells is 30% inhibited by 300 microM SITS. Characteristics of the SITS-sensitive flux pathway were examined in terms of sensitivity to changes in membrane potential and intracellular pH. Potential dependence was evaluated using unidirectional influx of [14C]tetraphenylphosphonium ([14C]-TPP+) as a qualitative sensor of diffusion potentials created by experimentally imposed gradients of Cl-. Steady-state distribution of [14C]methylamine ([14C]MA) was used to examine for Cl(-)-dependent changes in intracellular pH. Imposed Na+ gradients, but not Cl- gradients, induce changes in [14C]MA distribution. SITS does not alter the [14C]MA distribution observed in cells with imposed gradients of Na+ and Cl-. Both results suggest that inhibition of Cl(-)-OH- exchange system is not the basis for the SITS effect on Cl- influx. However, if relative permeabilities for ion pairs via conductance pathways are compared, it can be shown that SITS causes a marked reduction of Pcl relative to either PNa or PK. SITS also inhibits electrically induced influx of [14C]TPP+ or [14C] alpha-methylglucoside driven by imposed Cl- gradients. Conversely, electrically driven Cl- influx can be blocked by SITS. These observations are all consistent with a SITS-sensitive Cl- conductance pathway associated with the plasma membrane of chick intestinal cells. No Cl(-)-OH- exchange capability can be detected for chick intestinal cells.

High affinity L-aspartate transport in chick small intestine.

Epithelial cells isolated from chick small intestine were used to study the mechanism of L-aspartate transport. Two kinetically distinct uptake systems of high (Km' = 16 microM) and low (Km'' = 2.7 mM) affinity are observed. This paper examines the cation dependence and membrane potential sensitivity of the high affinity system. Unidirectional influx studies indicate that extracellular Na+ is an absolute requirement for transport function. Flux is optimal when K+ is present intracellularly, however this cation is not required for Na+-dependent L-aspartate uptake. In the absence of K+, flux enhancement is observed when the intracellular pH is acidic. In contrast, acidic intracellular pH is inhibitory in cells that are preequilibrated with K+. Sodium ([Na+]o greater than [Na+]i gradients, and potassium ([K+]o less than [K+]i) or proton ([H+]o less than [H+]i) gradients can independently energize the Na+-dependent accumulation of L-aspartate above equilibrium levels, suggesting that Na+ and L-aspartate cotransport occurs with concomitant K+ or H+ antiport. L-Aspartate influx is insensitive to membrane potential changes created by inwardly directed anion gradients in the presence or absence of intracellular K+. A model is presented that is consistent with electroneutral Na+-coupled transfer with an ion antiport site of low specificity.

Quantitative use of weak bases for estimation of cellular pH gradients.

An improvement in the usual procedure for estimating cellular H+ gradients by distribution of weak bases is described, which involves evaluation of and correction for the permeability of the ionic form of the sensor. In the case of methylamine, unidirectional uptake (influx) of the methylammonium ion is calculated by comparing the total influx of [14C]methylamine with the influx of the highly permeant ion [14C]tetraphenyl-phosphonium ([14C]TPP+) for two experimental situations in which the membrane potential differs. Comparison of the potential-dependent changes in unidirectional influx of methylamine and TPP+ allows calculation of the magnitude of influx for the methylammonium ion. This value can then be used to determine the error in the H+ gradient as estimated from the steady-state distribution of methylamine across the plasma membrane. By using ATP-depleted isolated small intestine cells from the chick as the test system, and imposed H+ gradients of defined magnitude, it can be shown that the observed error matches the calculated error.

Phlorizin binding to isolated enterocytes: membrane potential and sodium dependence.

Phlorizin binding is studied in isolated intestinal epithelial cells of the chick. Cells are ATP depleted to allow extensive manipulation of ionic gradients and membrane potential (delta psi). Phlorizin binding is assayed at steady state. Carrier specific phlorizin binding is defined as D-glucose (90 mM) inhibitable binding. Specific binding displays simple Michaelian kinetics as a function of phlorizin, indicating the presence of a single homogeneous binding site. Sodium concentrations and delta psi modify the apparent binding affinity but not the maximum number of binding sites. In contrast, the activation curve as a function of sodium concentrations is sigmoid and the apparent maximum number of binding sites at saturating sodium is phlorizin dependent. The rate of phlorizin association is both delta psi and sodium-concentration dependent. Dissociation is sodium-concentration dependent but not delta psi dependent. Theoretical analysis indicates binding order of substrates is random. In addition, data suggests that the phlorizin/sodium stoichiometry is 2:1. The delta psi dependence can be explained by two models: either translocation is the delta psi-dependent step and the free carrier is anionic, or sodium binding is the delta psi-dependent step.

A new method for determination of relative ion permeabilities in isolated cells.

The unidirectional influx of the lipophilic cation tetraphenylphosphonium (TPP+) into isolated epithelial cells is a function of the membrane potential that exists across the cellular plasma membrane. Because of the potential dependence, [14C]TPP+ influx can be used as a qualitative sensor of changes in the membrane potential induced by diffusion of ions after the experimental imposition of transmembrane ion gradients. This report describes a "crossover" procedure in which the influx of [14C]TPP+ during systematic changes in the ionic composition of incubation media is used to identify conditions in which no change in membrane potential occurs. The ion ratio at the crossover provides a measure of the relative permeabilities of the two test ions being compared. By using this approach, the ion permeabilities for intestinal epithelial cells prepared from White Rock chickens can be ranked relative to the permeability of Na+ (PNa), i.e., when PNa is equal to 1.0. The permeability sequence and relative values for ion permeability in this system are tris(hydroxymethyl)aminomethane-gluconate (less than 0.1) less than Li+ (0.3) less than Na+ (1.0) less than Cl- (2.0) less than K+ (6.0) = NO3- (6.0) less than SCN- (18) less than K+ + valinomycin (40). The procedure is general enough in principle to be of broad application to a wide variety of cell or membrane vesicle preparations.