Two mechanisms of H+/OH- transport across phospholipid vesicular membrane facilitated by gramicidin A.

Two rate-limiting mechanisms have been proposed to explain the gramicidin channel facilitated decay of the pH difference across vesicular membrane (delta pH) in the pH region 6-8 and salt (MCI, M+ = K+, Na+) concentration range 50-300 mM. 1) At low pH conditions (approximately 6), H+ transport through the gramicidin channel predominantly limits the delta pH decay rate. 2) At higher pH conditions (approximately 7.5), transport of a deprotonated species (but not through the channel) predominantly limits the rate. The second mechanism has been suggested to be the hydroxyl ion propogation through water chains across the bilayer by hydrogen bond exchange. In both mechanisms alkali metal ion transport providing the compensating flux takes place through the gramicidin channels. Such an identification has been made from a detailed study of the delta pH decay rate as a function of 1) gramicidin concentration, 2) alkali metal ion concentration, 3) pH, 4) temperature, and 5) changes in the membrane order (by adding small amounts of chloroform to vesicle solutions). The apparent activation energy associated with the second mechanism (approximately 3.2 kcal/mol) is smaller than that associated with the first mechanism (approximately 12 kcal/mol). In these experiments, delta pH was created by temperature jump, and vesicles were prepared using soybean phospholipid or a mixture of 94% egg phosphatidylcholine and 6% phosphatidic acid.     

The structure,cation binding,transport, and conductance of Gly15-gramicidin A incorporated into SDS micelles and PC/PG vesicles

To further investigate the effect of single amino acid substitution on the structure and function of the gramicidin channel, an analogue of gramicidin A (GA) has been synthesized in which Trp(15) is replaced by Gly in the critical aqueous interface and cation binding region. The structure of Gly(15)-GA incorporated into SDS micelles has been determined using a combination of 2D-NMR spectroscopy and molecular modeling. Like the parent GA, Gly(15)-GA forms a dimeric channel composed of two single-stranded, right-handed beta(6.3)-helices joined by hydrogen bonds between their N-termini. The replacement of Trp(15) by Gly does not have a significant effect on backbone structure or side chain conformations with the exception of Trp(11) in which the indole ring is rotated away from the channel axis. Measurement of the equilibrium binding constants and Delta G for the binding of monovalent cations to GA and Gly(15)-GA channels incorporated into PC vesicles using (205)Tl NMR spectroscopy shows that monovalent cations bind much more weakly to the Gly(15)-GA channel entrance than to GA channels. Utilizing the magnetization inversion transfer NMR technique, the transport of Na(+) ions through GA and Gly(15)-GA channels incorporated into PC/PG vesicles has been investigated. The Gly(15) substitution produces an increase in the activation enthalpy of transport and thus a significant decrease in the transport rate of the Na(+) ion is observed. The single-channel appearances show that the conducting channels have a single, well-defined structure. Consistent with the NMR results, the single-channel conductances are reduced by 30% and the lifetimes by 70%. It is concluded that the decrease in cation binding, transport, and conductance in Gly(15)-GA results from the removal of the Trp(15) dipole and, to a lesser extent, the change in orientation of Trp(11).     

23Na and 1H NMR studies on melittin channels activated by tricyclic tranquilizers.

A dynamic 23Na nuclear magnetic resonance (NMR) technique was applied to the exchange system of Na+ ions present inside and outside large unilamellar vesicles at an equivalent concentration. Addition of melittin to phosphatidylcholine vesicles did not induce any detectable Na+ transport across the membrane but subsequent addition of a trace of chlorpromazine or imipramine did induce Na+ transport. Because the formation of a drug-melittin adduct in a solution was detected by 1H NMR, the activation of melittin channels was assumed to originate from the direct interaction of the drug and melittin.     

Mechanisms of uptake of ketone bodies by luminal-membrane vesicles

The energetics and location of renal transport of acetoacetate, beta-hydroxybutyrate, alpha-hydroxybutyrate and gamma-hydroxybutyrate by luminal-membrane vesicles from either whole cortex or pars convoluta or pars recta of rabbit proximal tubule were studied. Addition of either acetoacetate or beta-hydroxybutyrate or its analogues to dye-membrane-vesicle suspensions in the presence of Na+ gradient (extravesicular greater than intravesicular) resulted in absorbance changes indicative of depolarizing event(s). Valinomycin enhanced the Na+-dependent uptake of monocarboxylic acids, provided a K+ gradient (intravesicular greater than extravesicular) was present. By contrast, Na+-dependent uptake of these compounds was nearly abolished by ionophores that permit Na+ to pass through the luminal-membrane via another channel, either electrogenically (e.g. gramicidin D) or electroneutrally (e.g. nigericin). These results established that the Na+-dependent transport of ketone bodies and analogues by luminal-membrane vesicles is an electrogenic process. Eadie-Hofstee analysis of saturation kinetic data suggested the presence of multiple transport systems in vesicles from whole cortex for these compounds. Tubular localization of the transport systems was studied by the use of vesicles derived from pars convoluta and pars recta. In pars recta uptake of all these compounds was mediated by means of a single high affinity common transport system. Uptake of these compounds by vesicles from pars convoluta was carried out via a relatively low affinity but common transport system. The physiological importance of the transport systems is discussed.     

Mechanism of imipramine inhibition of platelet 5-hydroxytryptamine transport.

Plasma membrane vesicles isolated from porcine blood platelets take up approximately 8 to 15 pmol of [3H]imipramine per mg of membrane protein. This apparent binding requires Na+ in the external medium and is reversed by 5-hydroxytryptamine and fluoxetine. The apparent KD for imipramine uptake is 23 nM, which agrees well with the KI for competitive inhibition of 5-hydroxytryptamine transport by imipramine. In contrast to 5-hydroxytryptamine transport, imipramine uptake is not dependent on transmembrane Na+ and K+ gradients and is insensitive to ionophores such as nigericin and gramicidin which dissipate these gradients. Although 5-hydroxytryptamine rapidly and competitively displaces imipramine from membrane vesicles, imipramine does not cause 5-hydroxytryptamine efflux and inhibits 5-hydroxytryptamine exchange. These results are consistent with the proposal that imipramine binds to the substrate site of the 5-hydroxytryptamine transporter but cannot be transported.     

Active amino acid transport in plasma membrane vesicles from Simian virus 40-transformed mouse fibroblasts. Characteristics of electrochemical Na+ gradient-stimulated uptake.

Selectively permeable membrane vesicles isolated from Simian virus 40-transformed mouse fibroblasts catalyzed Na+ gradient-coupled active transport of several neutral amino acids dissociated from intracellular metabolism. Na+-stimulated alanine transport activity accompanied plasma membrane material during centrifugation in discontinuous dextran 110 gradients. Carrier-mediated transport into the vesicle was demonstrated. When Na+ was equilibrated across the membrane, countertransport stimulation of L-[3H]alanine uptake occurred in the presence of accumulated unlabeled L-alanine, 2-aminoisobutyric acid, or L-methionine. Competitive interactions among neutral amino acids, pH profiles, and apparent Km values for Na+ gradient-stimulated transport into vesicles were similar to those previously described for amino acid uptake in Ehrlich ascites cells, which suggests that the transport activity assayed in vesicles is a component of the corresponding cellular uptake process. Both the initial rate and quasi-steady state of uptake were stimulated as a function of a Na+ gradient (external Na+ greater than internal Na+) applied artificially across the membrane and were independent of endogenous (Na+ + K+)-ATPase activity. Stimulation by Na+ was decreased when the Na+ gradient was dissipated by monensin, gramicidin D or Na+ preincubation. Na+ decreased the apparent Km for alanine, 2-aminoisobutyric acid, and glutamine transport. Na+ gradient-stimulated amino acid transport was electrogenic, stimulated by conditions expected to generate an interior-negative membrane potential, such as the presence of the permeant anions NO3- and SCN-. Na+-stimulated L-alanine transport was also stimulated by an electrogenic potassium diffusion potential (K+ internal greater than K+ external) catalyzed by valinomycin; this stimulation was blocked by nigericin. These observations provide support for a mechanism of active neutral amino acid transport via the "A system" of the plasma membrane in which both a Na+ gradient and membrane potential contribute to the total driving force.     

Sodium-dependent transport of L-leucine in membrane vesicles prepared from Pseudomonas aeruginosa.

Membrane vesicles were prepared by osmotic lysis of spheroplasts of Pseudomonas aeruginosa strain P14, and the active transport of amino acids was studied. D-Glucose, gluconate, and L-malate supported active transport of various L-amino acids. The respiration-dependent leucine transport was markedly stimulated by Na+. Moreover, without any respiratory substrate, leucine was also transported transiently by the addition of Na+ alone. This transient uptake of leucine was not inhibited either by carbonyl cyanide p-trifluoromethyoxyphenylhydrazone or by valinomycin, but was completely abolished by gramicidin D. Increase in the concentration of Na+ of the medium resulted in a decrease of the Km for L-leucine transport, whereas the Vmax was not significnatly affected. Active transport of leucine was inhibited competitively by isoleucine or by valine, whose transport was also stimulated by Na+. On the other hand, Na+ was not required for the uptake of other L-amino acids tested, but rather was inhibitory for some of them. These results show (i) that a common transport system for branched-chain amino acids exists in membrane vesicles, (ii) that the system requires Na+ for its activity, and (iii) that an Na+ gradient can drive the system.     

Active Na+-dependent transport of Ca 2+ into the fraction of smooth muscle sarcolemma vesicles

Studies on the vesicular fraction of myometrium sarcolemma showed that in the absence of initial Ca2+ gradient the vesicles activity accumulate Ca2+ by utilizing the energy of the antiport-directed Na+ gradient. Monensin (50 microM) suppresses practically completely the Ca2+ transport. The amount of Ca2+ entering the vesicles against the concentration gradient diminishes with a decrease in the oppositely directed Na+ gradient. Cd2+ (5 mM) causes a complete inhibition of active Ca2+ transport, whereas Mn2+ and Mg2+ inhibit this process by 85% and 35%, respectively; amiloride (500 microM) is fairly ineffective. In the absence of initial Ca2+ and Na+ gradients valinomycin (0.05-1 microM) does not affect the changes in Ca2+ concentration in the intravesicular volume both with and without K+ gradient. Under conditions of initial equilibrium for Ca2+ and Na+ the magnitude and sign of the membrane potential for the K(+)-valinomycin system have no effect on Ca2+ transport regardless of value of absolute Na+ concentration inside and outside the vesicles. Depolarization of membrane vesicles does not interfere with the Na(+)-driven active Ca2+ transport into the sarcolemma which is dependent on the energy of the Na+ gradient. Using calibration curves, it was shown that the physiologically significant (6-fold) Na+ gradient increases Ca2+ concentration in the intravesicular volume from 100 to 160-170 microM. Ac active potential-independent Ca2+ transport through the smooth muscle sarcolemma requires about one third (0.3 kcal/mol) of the Na+ gradient; energy the remainder is dissipated. It is concluded that in smooth muscles the Na+ gradient can provide the active transsarcolemmal transport of Ca2+.     

Ca2+-induced down-regulation of Na+ channels in toad bladder epithelium

Regulation of epithelial Na+ channels was investigated by measuring the amiloride-blockable 22Na+ fluxes in apical membrane vesicles, derived from cells exposed to various treatments. Maximal amiloride-blockable 22Na+ uptake into vesicles was obtained if the cells were preincubated at 25 degrees C in a Ca2+-free [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) solution. Including 10(-5) M Ca2+ in the cell incubating medium blocked nearly all of the amiloride-sensitive flux in vesicles, even though the Ca2+ was removed before homogenization of the cells. This Ca2+-dependent inhibition of Na+ channels could be induced in whole cells only; incubating cell homogenates with Ca2+ had no effect on the transport in vesicles. The dose-response relationships of this effect were measured by equilibrating cell aliquots with various Ca2+-EGTA buffers, preparing membrane vesicles (in the absence of Ca2+ ions), and assaying them for amiloride-sensitive Na+ permeability. It was found that the Ca2+ blockage is highly cooperative (Hill coefficient of nearly 4) and is characterized by an inhibition constant which varies between 6.4 X 10(-8) to 8.15 X 10(-6)M Ca2+. Thus, it is likely that the above process is involved in the physiological control of Na+ transport. The Ca2+-dependent transport changes were not affected by the calmodulin inhibitor trifluoperasine, vanadate (VO3-), phorbol ester, colchicine, cytochalasin B, 3-deazaadenosine, and 8-bromo-cAMP. Vanadyl (VO2+) ions, on the other hand, produced a "Ca2+-like" inhibition of transport.     

Stimulation of cation transport in mitochondria by gramicidin and truncated derivatives

Gramicidin and the truncated derivatives desformylgramicidin (desfor) and des(formylvalyl)gramicidin (desval) stimulate monovalent cation transport in rat liver mitochondria. Cation fluxes were compared indirectly from the effect of cations on the membrane potential at steady state (state 4) or from the associated stimulation of electron transport. Rb+ transport was measured directly from the uptake of 86Rb. The truncated gramicidins show enhanced selectivity for K+ and Rb+ when compared to gramicidin. Moreover, the pattern of selectivity within the alkali cation series is altered, i.e., Rb+ greater than K+ greater than Cs+ greater than Na+ greater than Li+ for desfor and desval as compared to Cs+ greater than Rb+ greater than K+ = Na+ greater than Li+ for gramicidin. The cation fluxes through the truncated derivatives are more strongly dependent on the cation concentration. The presence of high concentrations of permeating cation enhances the transport of other cations through the truncated derivative channels, suggesting that cations are required for stabilizing the channel structure. In high concentrations of KCl, desfor and desval are nearly as effective as gramicidin in collapsing the mitochondrial membrane potential, and, consequently, in the uncoupling of oxidative phosphorylation and enhancement of ATP hydrolysis. Preliminary experiments with liposomes show that 86Rb exchange is stimulated by desfor and desval almost to the same extent as gramicidin. These results strongly suggest that the truncated gramicidins form a novel conducting channel which differs from the gramicidin head-to-head, single-stranded beta 6.3-helical dimer ("channel") in its conductance characteristic and its structure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estimation of Na+ dwell time in the gramicidin A channel. Na+ ions as blockers of H+ currents

The permeation of Na+ through gramicidin A channels shows a simple saturation with increasing Na+ concentration that can be described by two different models. The first model assumes that one Na+ binds to the channel with high affinity (approximately 30 M-1) and that conduction occurs by a 'knock-on' mechanism requiring double occupancy of the channel; the other model assumes that Na+ binding is of low affinity (less than 1 M-1), and that double occupancy of the channel is rare. NMR measurements have shown tight Na+ binding, favoring the first model, but measurements of flux ratios and water transport support the second model. We present here a relatively model-independent measurement of the dwell time of Na+ inside the channel, in which we characterize the fluctuations in H+ current through the channel induced by 'block' from the more slowly permeating Na+ ions. The mean Na+ dwell time inside the channel is estimated to be approximately 10 ns at a membrane potential of 200 mV. This result is inconsistent with tight Na+ binding, thus favoring the second model.     

Sodium gradient-dependent L-glutamate transport is localized to the canalicular domain of liver plasma membranes. Studies in rat liver sinusoidal and canalicular membrane vesicles

The driving forces for L-glutamate transport were determined in purified canalicular (cLPM) and basolateral (i.e. sinusoidal and lateral; blLPM) rat liver plasma membrane vesicles. Initial rates of L-glutamate uptake in cLPM vesicles were stimulated by a Na+ gradient (Na+o greater than Na+i), but not by a K+ gradient. Stimulation of L-glutamate uptake was specific for Na+, temperature sensitive, and independent of nonspecific binding. Sodium-dependent L-glutamate uptake into cLPM vesicles exhibited saturation kinetics with an apparent Km of 24 microM, and a Vmax of 21 pmol/mg X min at an extravesicular sodium concentration of 100 mM. Specific anionic amino acids inhibited L-[3H]glutamate uptake and accelerated the exchange diffusion of L-[3H]glutamate. An outwardly directed K+ gradient (K+i greater than K+o) further increased the Na+ gradient (Na+o greater than Na+i)-dependent uptake of L-glutamate in cLPM vesicles, resulting in a transient accumulation of L-glutamate above equilibrium values (overshoot). The K+ effect had an absolute requirement for Na+. In contrast, in blLPM the initial rates of L-glutamate uptake were only minimally stimulated by a Na+ gradient, an effect that could be accounted for by contamination of the blLPM vesicles with cLPM vesicles. These results indicate that hepatic Na+ gradient-dependent transport of L-glutamate occurs at the canalicular domain of the plasma membrane, whereas transport of L-glutamate across sinusoidal membranes results mainly from passive diffusion. These findings provide an explanation for the apparent discrepancy between the ability of various in vitro liver preparations to transport glutamate and suggest that a canalicular glutamate transport system may serve to reabsorb this amino acid from bile.     

Conformational transitions in fluorescein-labeled (Na,K)ATPase reconstituted into phospholipid vesicles

Fluorescein-labeled (Na,K)ATPase reconstituted into phospholipid vesicles has been used to study conformational transitions. Addition of K+ or Na+ to the vesicle medium induces fluorescence changes characteristic of the E2(K) or E1Na states of fluorescein-labeled (Na,K)ATPase (Karlish, S.J.D. (1980) J. Bioenerg. Biomembr. 12, 111-136). The cation effects are exerted from the cytoplasmic surface of inside-out-oriented pumps. Equilibrium cation titrations and measurements of rates of conformational transitions have led to the following observations. 1) The rate of E2(K)----E1Na or E2(T1)----E1Na is 4-6-fold faster and E1K----E2(K) is about 2-fold slower in vesicles compared to enzyme. In equilibrium titrations the K0.5 for K+ is higher and that for Na+ is lower for vesicles compared to enzyme. The conformational equilibrium E(1)2K----E2(2K) is apparently shifted toward E(1)2K in vesicles compared to enzyme. 2) Diffusion potentials, positive-outside, induced with valinomycin or Li+ ionophore AS701, do not affect the rates of E2(T1)----E1Na or E1K----E2(K), or equilibrium cation titrations. This demonstrates that the conformational transitions E(1)2K----E2(2K) are voltage-insensitive steps, confirming a prediction based on transport experiments. 3) In vesicles containing choline, K+, Na+, or Li+, the rate of E2(T1)----E1Na increases in the order given. Vesicles with reconstituted fluorescein-labeled (Na,K)ATPase provide a convenient system for correlating directly properties of conformational transitions with cation transport.     

L-glutamate stimulation of Na+ efflux from brain synaptic membrane vesicles

The characteristics of 22Na efflux from 22NaCl-preloaded synaptic plasma membrane vesicles and the stimulation of such efflux by gramicidin D and L-glutamate were determined. The rate and magnitude of passive Na+ efflux were dependent on the initial intravesicular NaCl concentration. A Na+:cation exchange process was also observed. Gramicidin D markedly enhanced Na+ efflux in a concentration-dependent manner and at 10 microM it caused total loss of intravesicular 22Na. The neuroexcitatory amino acids L-glutamate and D-glutamate, and the amino acid analog kainic acid, also stimulated Na+ efflux in a dose-dependent fashion, but their effects were weaker than those of gramicidin D. The mechanism of glutamate stimulation of Na+ flux is presumed to be through the activation of the glutamate receptor . Na+ channel complex in these membranes.     

K+-independent active transport of Na+ by the (Na+ and K+)-stimulated adenosine triphosphatase

The (Na+ and K+)-stimulated adenosine triphosphatase (Na+,K+)-ATPase) from canine kidney reconstituted into phospholipid vesicles showed an ATP-dependent, ouabain-inhibited uptake of 22Na+ in the absence of added K+. This transport occurred against a Na+ concentration gradient, was not affected by increasing the K+ concentration to 10 microM (four times the endogenous level), and could not be explained in terms of Na+in in equilibrium Na+out exchange. K+-independent transport occurred with a stoichiometry of 0.5 mol of Na+ per mol of ATP hydrolyzed as compared with 2.9 mol of Na+ per mol of ATP for K+-dependent transport.     

Peroxyl radicals promoted changes in water permeability through gramicidin channels in DPPC and lecithin-PC vesicles

Gramicidin incorporation to DPPC or lecithin-PC large unilamellar vesicles (LUVs) leads to pore formation that, under hyper-osmotic conditions, produces a noticeable increase in the rate of trans-membrane water flow. This pore formation is more efficient in the more fluid lecithin-PC LUVs. Exposure of these vesicles to peroxyl radicals generated in the aerobic thermolysis of 2,2'-azo-bis(2-amidinopropane) (AAPH), changes the physical properties of the bilayer (as sensed employing fluorescent probes), modifies gramicidin molecules (as sensed by the decrease in Trp fluorescence) and notably reduces the transbilayer rate of water outflow. In order to evaluate if this reduced water-transport capacity is due to changes in the membrane due to lipid-peroxidation and/or direct damage to gramicidin channels, results obtained in the oxidable vesicles (lecithin-PC) were compared to those obtained in DPPC vesicles. The data obtained show that most of the water transport efficiency loss can be ascribed to a direct disruption of gramicidin channels by AAPH derived peroxyl radicals.     

Single-channel studies on linear gramicidins with altered amino acid side chains. Effects of altering the polarity of the side chain at position 1 in gramicidin A.

The modulation of gramicidin A single-channel characteristics by the amino acid side chains was investigated using gramicidin A analogues in which the NH2 terminal valine was chemically replaced by other amino acids. The replacements were chosen such that pairs of analogues would have essentially isosteric side chains of different polarities at position 1 (valine vs. trifluorovaline or hexafluorovaline; norvaline vs. S-methyl-cysteine; and norleucine vs. methionine). Even though the side chains are not in direct contact with the permeating ions, the single-channel conductances for Na+ and Cs+ are markedly affected by the changes in the physico-chemical characteristics of the side chains. The maximum single-channel conductance for Na+ is decreased by as much as 10-fold in channels formed by analogues with polar side chains at position 1 compared with their counterparts with nonpolar side chains, while the Na+ affinity is fairly insensitive to these changes. The relative conductance changes seen with Cs+ were less than those seen with Na+; the ion selectivity of the channels with polar side chains at position 1 was increased. Hybrid channels could form between compounds with a polar side chain at position 1 and either valine gramicidin A or their counterparts with a nonpolar side chain at position 1. The structure of channels formed by the modified gramicidins is thus essentially identical to the structure of channels formed by valine gramicidin A. The polarity of the side chain at position 1 is an important determinant of the permeability characteristics of the gramicidin A channel. We discuss the importance of having structural information when interpreting the functional consequences of site-directed amino acid modifications.     

Functional differences of Na+/Ca2+ exchanger expression in Ca2+ transport system of smooth muscle of guinea pig stomach

The plasma membrane ATP-dependent Ca2+ pump and the Na+/Ca2+ exchanger (NCX) are the major means of Ca2+ extrusion in smooth muscle. However, little is known regarding distribution and function of the NCX in guinea pig gastric smooth muscle. The expression pattern and distribution of NCX isoforms suggest a role as a regulator of Ca2+ transport in cells. Na+ pump inhibition and the consequent to removal of K+ caused gradual contraction in fundus. In contrast, the response was significantly less in antrum. Western blotting analysis revealed that NCX1 and NCX2 are the predominant NCX isoforms expressed in stomach, the former was expressed strongly in antrum, whereas the latter displayed greater expression in fundus. Isolated plasma membrane fractions derived from gastric fundus smooth muscle were also investigated to clarify the relationship between NCX protein expression and function. Na+-dependent Ca2+ uptake increased directly with Ca2+ concentration. Ca2+ uptake in Na+-loaded vesicles was markedly elevated in comparison with K+-loaded vesicles. Additionally, Ca2+ uptake by the Na+- or K+-loaded vesicles was substantially higher in the presence of A23187 than in its absence. The result can be explained based on the assumption that Na+ gradients facilitate downhill movement of Ca2+. Na+-dependent Ca2+ uptake was abolished by the monovalent cationic ionophore, monensin. NaCl enhanced Ca2+ efflux from vesicles, and this efflux was significantly inhibited by gramicidin. Results documented evidence that NCX2 isoform functionally contributes to Ca2+ extrusion and maintenance of contraction-relaxation cycle in gastric fundus smooth muscle.     

The monensin-mediated transport of sodium ions through phospholipid bilayers studied by 23Na-NMR spectroscopy

The monensin-mediated transport of sodium ions through the walls of large unilamellar vesicles of egg phosphatidylcholine was studied using 23Na-NMR and aqueous shift reagents. The transport is dynamic on the NMR time-scale and is strictly first order in monensin over the concentration ranges studied indicating that transport occurs by a 1:1 Na+-ionophore complex. Transport appears to be inhibited by increasing concentrations of Na+.     

Properties of gramicidin A channels in erythrocyte membranes

Studies for the cation permeability properties of the gramicidin A channel in erythrocyte membranes are presented. It is shown that gramicidin A interacts with the membrane in a cooperative manner, creating aggregates of the antibiotic molecules in the lipid lattice of the membrane. Cationic channels exist in these aggregates with the following order of selectivity: Rb+ greater than Cs+ greater K+ greater than Na+. The cation permeability of the channels depends on the media surrounding the membrane. This finding has been explained on the basis of Hodgkin-Keynes theory for single-file ion diffusion through extra-narrow pores.