Ionic permeability of K, Na, and Cl in potassium-depolarized nerve. Dependency on pH, cooperative effects, and action of tetrodotoxin.

The passive ionic membrane conductances (gj) and permeabilities (Pj) of K, Na, and Cl of crayfish (Procambarus clarkii) medial giant axons were determined in the potassium-depolarized axon and compared with that of the resting axon. Passive ionic conductances and permeabilities were found to be potassium dependent with a major conductance transition occurring around an external K concentration of 12-15 mM (Vm = -60 to -65 mV). The results showed that K, Na, and Cl conductances increased by 6.2, 6.9, and 27-fold, respectively, when external K was elevated from 5.4 to 40 mM. Permeability measurements indicated that K changed minimally with K depolarization while Na and Cl underwent an order increase in permeability. In the resting axon (K0 = 5.4 mM, pH = 7.0) PK = 1.33 X 10(-5), PCl = 1.99 X 10(-6), PNa = 1.92 X 10(-8) while in elevated potassium (K0 = 40 mM, pH 7.0), PK = 1.9 X 10(-5), PCl = 1.2 X 10(-5), and PNa = 2.7 X 10(-7) cm/s. When membrane potential is reduced to 40 mV by changes in internal ions, the conductance changes are initially small. This suggests that resting channel conductances depend also on ion environments seen by each membrane surface in addition to membrane potential. In elevated potassium, K, Na, and Cl conductances and permeabilities were measured from pH 3.8 to 11 in 0.2 pH increments. Here a cooperative transition in membrane conductance or permeability occurs when pH is altered through the imidazole pK (approximately pH 6.3) region. This cooperative conductance transition involves changes in Na and Cl but not K permeabilities. A Hill coefficient n of near 4 was found for the cooperative conductance transition of both the Na and Cl ionic channel which could be interpreted as resulting from 4 protein molecules forming each of the Na and Cl ionic channels. Tetrodotoxin reduces the Hill coefficient n to near 2 for the Na channel but does not affect the Cl channel. In the resting or depolarized axon, crosslinking membrane amino groups with DIDS reduces Cl and Na permeability. Following potassium depolarization, buried amino groups appear to be uncovered. The data here suggest that potassium depolarization produces a membrane conformation change in these ionic permeability regulatory components. A model is proposed where membrane protein, which forms the membrane ionic channels, is oriented with an accessible amino terminal group on the axon exterior. In this model the ionizable groups on protein and phospholipid have varied associations with the different ionic channel access sites for K, Na, and Cl, and these groups exert considerable control over ion permeation through their surface potentials.     

Membrane potential measurements in isolated rat liver plasma membrane vesicles: effect of transmembrane ion concentration gradients

In isolated basolateral and canalicular rat liver plasma membrane vesicles the membrane potential (measured with DiS-C2 (5] varied with transmembrane concentration gradients of Na+, K+ and Cl- revealing the following ion permeabilities: basolateral vesicles: PNa/PK: 0.76, PCl/PK: 0.45 and canalicular vesicles: PNa/PK: 0.69, PCl/PK: 0.56. The data indicate a permselectivity of PK greater than PNa greater than PCl for both membranes.     

The electrical potential profile of gallbladder epithelium.

In this study the relative ionic permeabilities of the cell membranes of Necturus gallbladder epithelium have been determined by means of simultaneous measurement of transmural and transmucosal membrane potential differences (PD) and by ionic substitution experiments with sodium, potassium and chloride ions. It is shown that the mucosal membrane is permeable to sodium and to potassium ions. The baso-lateral membrane PD is only sensitive to potassium ions. In both membranes chloride conductance is negligible or absent. The ratio of the resistances of the mucosal and baso-lateral membranes, RM/RS, increases upon reducing the sodium concentration in the mucosal solution. The same ratio decreases when sodium is replaced by potassium which implies a greater potassium than sodium conductance in the mucosal membrane. The relative permeability of the shunt for potassium, sodium and chloride ions is: PK/PNa/PCl=1.81:1.00:0.32. From the results obtained in this study a value for the PK/PNa ratio of the mucosal membrane could be evaluated. This ratio is 2.7. From the same data the magnitude of the electromotive forces generated across the cell membranes could be calculated. The EMF's are -15mV across the mucosal membrane and -81mV across the baso-lateral one. Due to the presence of the low resistance shunt the transmucosal membrane PD is -53.2mV (cell inside negative) and the transmural PD is +2.6mV (serosal side positive). The change in potential profile brought about by the low resistance shunt favors passive entry of Na ions into the cell across the mucosal membrane. Calculations show that this passive Na influx is maximally 64% of the net Na flux estimated from fluid transport measurements. The C-1 conductive of the baso-lateral membrane is too small to allow electrogenic coupling of C1 with Na transport across this membrane. Experiments with rabbit gallbladder epithelium indicate that the membrane properties in this tissue are qualitatively similar to those of Necturus gallbladder epithelium.     

Electrical resistance of muscle capillary endothelium.

A recently developed technique for in vivo determination of the electrical resistance of vascular endothelium in microvessels was applied to the vessels in a thin frog muscle, m. cutaneus pectoris. The technique consists of injection of current via a glass micropipette into a capillary and measurement of the resulting intra- and extravascular potential profiles with another micropipette placed at various distances from the current source. The theory of Peskoff and Eisenberg (1974) was used to handle the problems arising from distributed extravascular resistances and was experimentally shown to describe the external field satisfactorily. With this extension of one-dimensional cable theory the specific electrical resistance of arterial microvessels was 33 omega cm2 and of venous capillaries 23 omega cm2. The "length constants" were 135 and 112 micrometers, respectively. If results from arterial and venous vessels are taken together, the ionic permeabilities at 20 degrees C were PNa = 3.9 X 10(-5) cm X s-1, PK = 5.7 X 10(-5) cm X s-1, PCl = 5.9 X 10(-5) cm X s-1 and PHCO3 = 3.4 X 10(-5) cm X s-1. These figures agree with figures for capillary permeability obtained in tracer experiments on whole muscle. The study bridges a gap between single capillary and whole organ techniques with the conclusion that the two different approaches lead to similar results in muscle capillaries.     

Transmembrane K+, Na+, and Cl- permeability and conductance changes in the frog sartorius fibres following ouabain and zero [K+]o treatment

Changes in the K+, Na+, and Cl- permeabilities (P) and conductances (g) of the intact frog sartorius fibre membrane following ouabain or zero [K+]o treatment were calculated from intrafibre activity and whole muscle electrolyte changes. Conventional equations relating ionic fluxes to resting potential (E), ionic gradient potential, and internal and external ionic activities were used. Both treatments produced a three- to five-fold increase in PNa and gNa. In addition, ouabain produced a fivefold increase in PK (and gK) and a small decrease in PCl (and gCl), whereas zero [K+]o produced a 60% reduction in PK, a 90% reduction in gK, and a threefold increase in PCl (and gCl). When the two treatments were combined, the P and g changes were paradoxical, suggesting that the ouabain-induced increase in gK and the zero [K+]o-induced decrease in gK were occurring but in different channels (or carriers). During ouabain treatment, E reflects mainly the transmembrane K+ gradient potential; during zero [K+]o treatment, E reflects mainly the Cl- gradient potential. Despite channel (or carrier) specificity, it appears that all three ionic permeabilities are altered during the perturbations.     

Electrical properties of normal and transformed mammalian cells.

The transmembrane potential difference, Em, and DC membrane resistance were measured in 3T3 and polyoma virus-transformed 3T3 cells. Em was a function of cell density and was -12 and -25 mV for the normal and transformed cells, respectively. The external concentrations of K+, Na+, and Cl were varied in order to study the nature of the differences between the two cell types. The relative permeability of ions was calculated to be: PNa/PK, 1.0; PCl/PK, 1.88; PNa/PCl, 0.53 for 3T3 cells, and 0.27, 1.75, and 0.15 for the transformed cells. In contrast to the normal cells, PNa/PK varied as a function of the external K+ concentration for the transformed cells. It was emphasized that the manipulation of variables directly affecting the electrical properties of cells also involves the indirect manipulation of a network of interconnected physiological determinants.     

Cation and anion sequences in dark-adapted Balanus photoreceptor.

Anion and cation permeabilities in dark-adapted Balanus photoreceptors were determined by comparing changes in the membrane potential in response to replacement of the dominant anion (Cl-) or cation (Na+) by test anions or cations in the superfusing solution. The anion permeability sequence obtained was PI greater than PSO4 greater than PBr greater than PCl greater than Pisethionate greater than Pmethanesulfonate. Gluconate, glucuronate, and glutamate generally appeared more permeable and propionate less permeable than Cl-. The alkali-metal cation permeability sequence obtained was PK greater than PRb greater than PCx greater than PNa approximately PLi. This corresponds to Eisenman's IV which is the same sequencethat has been obtained for other classes of nerve cells in the resting state. The values obtained for the permeability ratios of the alkali-metal cations are considered to be minimal. The membrane conductance measured by passing inward current pulses in the different test cations followed the sequence, GK greater than GRb greater than GCs greater than GNa greater than GLi. The conductance ratios obtained for a full substitution of the test cation agreed quite well with permeability ratios for all the alkali-metal cations except K+ which was generally higher.     

Explanation for constancy of intracellular chloride concentration with respect to membrane potential in guinea pig mammary gland.

1. The relative permeabilities for sodium, potassium and chloride in guinea pig mammary gland slices are determined by means of ion flux studies with radioisotopes. 2. Assuming that there are no significant electrogenic potential components, we calculate permeability ratios PNa/PK = 0.97 and Pc1/PK = 1.25. 3. Substitution of these values in the Goldman equation yields membrane potentials of--15 mV before and--13 mV after ouabain treatment. 4. This small change in membrane potential explains the absence of a significant change in chloride content upon ouabain application, which leads to large changes in intracellular sodium and potassium concentrations.     

Regulation of ion permeabilities of isolated rat liver cells by external calcium concentration and temperature.

Regulation of ion transport through the plasma membrane was studied on single cell suspensions of hepatocytes, obtained after perfusion of rat liver with collagenase/hyaluronidase solution. Steady-state intracellular K and Na contents were shown to be markedly dependent on external Ca concentration and temperature, the sum of both ion concentrations remaining nearly constant. In contrast, steady-state intracellular chloride content was found to be independent of external Ca concentration, but dependent on temperature. Using the constant field relations, the passive permeabilities PK and PCl for potassium and chloride, respectively, were derived from the experimental data. At temperatures at and above 37 degrees C, with increasing external Ca concentration, PK, exhibits a sharp decrease at about 10(-4)M. In contrast, PCl at 37 degrees C was found to be independent of Ca concentration within experimental error. Earth alkali ions other than Ca, show marked but different effects on PK if compared at equal concentrations. Preincubation of the cells with cholesterol leads to a broadening of the dependence of PK on external Ca concentration. The above results, as well as those on the dependence of PK on external Ca concentration obtained by other authors, could be quantitatively described by a theoretical model of the plasma membrane proposed earlier. This model postulates regulatory binding sites, which cooperatively undergo a cation exchange of divalent cations by K+ ions from the external medium if the cation composition of the latter is altered.     

Current-voltage relationship of the basolateral membrane of a tight epithelium.

The polyene antibiotic nystatin is used to reduce selectively to zero the apical membrane resistance of the rabbit descending colon, allowing the measurement of the current-voltage curve of the basolateral membrane. The I--V relationship is described by the Goldman-Hodgkin-Katz equations allowing calculation of PNa/PK, PCl/PK and PK for the basolateral membrane. Cs+ is found to block inward current (serosa to mucosa) in a manner similar to that found in excitable membranes.     

Effect of Salt Stress on Ion Channel Selective Permeability of Plasmalemma in Sorghum Roots

100 mmol/L, 200 mmol/L and 300 mmol/L NaC1 was used in proper order to treat three-day old seedlings of Sorghum vulgare Pets. The plasma membrane of roots was isolated and purified by aqueous biphasic partition device. The plasmalemma was incorporated into planar bilayer lipid membrane and ion channels were measured by electrical methods. Ion selective permeabilities (PK: PNa) were assayed in asymmetrical solutions containing 100 mmol/L NaC1 in Cis chamber and 100 mmol/L KC1 in Tran with chamber and were calculated from Goldman-Hodgkin-Kaltz equation. PK: PNa was 3.5 in plasmalemma of control roots and 1.5 in plasmalemma of salt stressed roots. It showed that the changes of ion selective permeability was very significant under salt stress. The importance of the change of ion selective permeability is discussed.     

Effect of oxytocin on the electrical potential and ion permeability of the apical membrane of frog gall bladder epithelial cells

The influence of oxytocin on the intracellular Na+ and K+ concentrations, the level of transmembrane potential differences, and on the relative ionic permeability (PNa/PK) of the apical zones of the superficial epithelium membrane was studied in experiments on the isolated frog gallbladder (GB). Oxytocine introduced into the outer incubation solution in a dose of 20 mulliunits/ml caused a reduction of transmembrane potential difference, and an increase of PNa/pk coefficient and an insignificant shift of the Na+ and K+ concentrations in the intracellular medium. Thirty minutes after the oxytocine action of the organ the membrane potential (MP) of the cells decreased from 52.7 mV to 38.7 mV (the cell is negatively charged inside), and PNa/PK increased from 0,083 (control) to 0,175 (test) with a simultaneous increase in the intracellular Na+ concentration by 18.3 milliequiv./kg of (H2O)i. Such a shift in the intracellular Na+ and K+ concentrations may cause a decrease of the MP by only--0.7 mV, but actually the membrane potential decreased by--14.0 mV. Thus, the reduction of the transmembrane potential difference results from increase of PNa/PK under the influence of oxytocine. No electrogenic ionic transport through the apical membrane of frog gallbladder epithelial cells was revealed.     

Sodium channel selectivity. Dependence on internal permeant ion concentration

The selectivity of sodium channels in squid axon membranes was investigated with widely varying concentrations of internal ions. The selectivity ratio, PNa/PK, determined from reversal potentials decreases from 12.8 to 5.7 to 3.5 as the concentration of internal potassium is reduced from 530 to 180 to 50 mM, respectively. The internal KF perfusion medium can be diluted by tetramethylammonium (TMA), Tris, or sucrose solutions with the same decrease in PNa/PK. The changes in the selectivity ratio depend upon internal permeant ion concentration rather than ionic strength, membrane potential, or chloride permeability. Lowering the internal concentration of cesium, rubidium, guanidnium, or ammonium also reduces PNa/Pion. The selective sequence of the sodium channel is: Na greater than guanidinium greater than ammonium greater than K greater than Rb greater than Cs.     

Chloride movements in human neutrophils. Diffusion, exchange, and active transport

Chloride content and fluxes were measured in isolated resting human peripheral polymorphonuclear leukocytes. The intracellular Cl concentration of cells kept at 37 degrees C in 148 mM Cl media was approximately 80 meq/liter cell water, fourfold higher than expected for passive distribution at the cell's estimated membrane potential (approximately -53 mV). All intracellular Cl was rapidly exchangeable with external 36Cl. Cells lost Cl exponentially into Cl-free media, and reaccumulated it when Cl was restored to the bath; this reuptake was dependent on metabolism. One-way 36Cl fluxes in steady state cells were approximately 1.4 meq/liter X min. The bulk (approximately 70%) of these represented electrically silent Cl/Cl exchange mediated by a carrier insensitive to disulfonic stilbenes but blocked by the anion carrier inhibitor alpha-cyano-4-hydroxycinnamate (CHC). The remaining fluxes were characterized in some detail. About 20% of 36Cl influx behaved as active transport: it moved thermodynamically uphill and was absent in cells treated with 2-deoxy-D-glucose, displayed Michaelis-Menten kinetics with Km(Cl) congruent to 5 mM, Vmax congruent to 0.25 meq/liter X min, and was inhibited by CHC (Ki congruent to 1.7 mM), ethacrynate (Ki congruent to 50 microM), and furosemide (Ki congruent to 50 microM). About 30% of Cl efflux and approximately 8% of Cl influx behaved as electrodiffusion through a low-permeability pathway (PCl congruent to 4 X 10(-9) cm/s; gCl congruent to 1 microsecond/cm2; PK/PNa/PCl congruent to to 10:1:1); these fluxes were linear with concentration and strongly voltage sensitive. The putative Cl channel does not appear to be voltage gated, and gives evidence of single filing.     

Ion transport by rabbit colon. I. Active and passive components.

Descending rabbit colon, stripped of muscularis externa, absorbs Na and Cl under short-circuit conditions and exhibits a residual ion flux, consistent with HCO3 secretion, whose magnitude is approximately equal to the rate of active Cl absorption. Net K transport was not observed under short-circuit conditions. The results of ion replacement studies and of treatment with ouabain or amiloride suggest that the short-circuit current ISC is determined solely by the rate of active Na transport and that the net movements of Cl and HCO3 are mediated by a Na-independent, electrically-neutral, anion exchange process. Cyclic AMP stimulates an electrogenic Cl secretion, abolishes HCO3 secretion but does not affect the rate of Na absorption under short-circuit conditions. Studies of the effect of transepithelial potential difference on the serosa-to-mucosa fluxes Jism of Na, K and Cl suggest that JNasm,JIsm and one-third of JCl-sm may be attributed to ionic diffusion. The permeabilities of the passive conductance pathway(s) are such that Pk:PNa:PCl= 1.0:0.07:0.11. Electrolyte transport by in vitro rabbit colon closely resembles that reported from in vivo studies of mammalian colon and thus may serve as a useful model for the further study of colonic ion transport mechanisms.     

Lithium absorption in tight and leaky segments of intestine

There is significant absorption of Li+ by human jejunum and ileum, but negligible absorption by human colon. Thus, a proximal-to-distal gradient of decreasing Li+ absorption and increasing junctional tightness exists in intestine as well as in renal tubule. For six leaky epithelia the relative permeabilities of K+, Na+, and Li+ by the junctional route are in the sequence PK greater than PNa greater than PLi and all fall within a factor of 2.5. In contrast, for tight epithelia PLi approximately PNa much greater than PK in the amiloride-sensitive channel of the apical membrane, but PK much greater than PLi approximately PNa in the basolateral membrane. The ability of several tight epithelia to sustain nonzero transepithelial Li+ absorption despite this basolateral barrier may be due to Na+/Li+ countertransport at the basolateral membrane, resulting in secondary active transport of Li+ across the epithelium.     

Ca2+-activated K+ channels in human red cells. Comparison of single-channel currents with ion fluxes

Exposure of the inner surface of intact red cells or red cell ghosts to Ca2+ evokes unitary currents that can be measured in cell-attached and cell-free membrane patches. The currents are preferentially carried by K+ (PK/PNa 17) and show rectification. Increasing the Ca2+ concentration from 0 to 5 microM increases the probability of the open state of the channels parallel to the change of K+ permeability as observed in suspensions of red cell ghosts. Prolonged incubation of red cell ghosts in the absence of external K+ prevents the Ca2+ from increasing K+ permeability. Similarly, the probability to find Ca2+-activated unitary currents in membrane patches is drastically reduced. These observations suggest that the Ca2+-induced changes of K+ permeability observed in red cell suspensions are causally related to the appearance of the unitary K+ currents. Attempts to determine the number of K+ channels per cell were made by comparing fluxes measured in suspensions of red cells with the unitary currents in membrane patches as determined under comparable ionic conditions. At 100 mM KCl in the external medium, where no net movements of K+ occur, the time course of equilibration of 86Rb+ does not follow a single exponential. This indicates a heterogeneity of the response to Ca2+ of the cells in the population. The data are compatible with the assumption that 25% of the cells respond with Pk = 33.2 X 10(-14)cm3/s and 75% with Pk = 3.1 X 10(-14)cm3/s. At 100 mM external K+ the zero current permeability of a single channel is 6.1 X 10(-14)cm3/s (corresponding to a conductance of 22 pS).(ABSTRACT TRUNCATED AT 250 WORDS)     

Arg352 is a major determinant of charge selectivity in the cystic fibrosis transmembrane conductance regulator chloride channel

The cystic fibrosis transmembrane conductance regulator forms an anion-selective channel. We previously showed that charge selectivity, the ability to discriminate between anions and cations, occurs near the cytoplasmic end of the channel. The molecular determinants of charge selectivity, however, are unknown. We investigated the role of Arg352, a residue flanking the predicted cytoplasmic end of the M6 segment, in the mechanism of charge selectivity. We determined the Cl- to Na+ permeability ratio (PCl/PNa) from the reversal potential measured in a 10-fold NaCl gradient. For the wild type, PCl/PNa was 36 (range of 28-51). For the R352H mutant, PCl/PNa was dependent on cytoplasmic pH. At pH 5.4, the PCl/PNa was 33 (range of 27-41), similar to that of the wild type, but at pH 7.2, where the histidine should be largely uncharged, PCl/PNa was 3 (range of 2.9-3.1). For the R352C and R352Q mutants, PCl/PNa was 7 (range of 6-8) and 4 (range of 3.5-4.4), respectively. Furthermore, Na+ which does not carry a significant fraction of the current through the wild type is measurably conducted through R352Q. Thus, the charge of the side chain at position 352 is a strong determinant of charge selectivity. In the wild type, the positive charge on Arg352 contributes to an electrostatic potential in the channel that forms a barrier to cation permeation. Mutation of Arg352 did not alter the halide selectivity sequence. Selectivity among halides must involve other residues.     

Ion selectivity of aqueous leaks induced in the erythrocyte membrane by crosslinking of membrane proteins

The aqueous leak induced in the human erythrocyte membrane by crosslinking of spectrin via disulfide bridges formed in the presence of diamide (Deuticke, B., Poser, B., Lütkemeier, P. and Haest, C.W.M. (1983) Biochim. Biophys. Acta 731, 196-210) was further characterized with respect to its ion selectivity by means of (a) measurements of cell volume changes or hemolysis, (b) determination of membrane potentials and (c) analysis of potential-driven ion fluxes. The leak turned out to be slightly cation-selective (PK:PCl approximately equal to 4:1). It discriminates mono- from divalent ions (PNa:PMg greater than 100:1, PCl:PSO4 greater than 10:1) and to a much lesser extent monovalent ions among each other. The selectivities for monovalent ions follow the sequence of free solution mobilities, increasing in the order Li+ less than or equal to Na+ less than K+ less than or equal to Rb+ less than Cs+ and F- less than Cl- less than Br- less than I-. Polyatomic anions also fit into that order. Quantitatively, the ratios of permeabilities of the leak are larger than those of the ion mobilities in free solution. The ion permeability of the leak is concentration-independent up to at least 150 mM. The ion milieu, however, has marked effects on leak permeability, most pronounced for chaotropic ions (guanidinium, nitrate, thiocyanate), which increase leak fluxes of charged and uncharged solutes. The results support the view that, besides geometric constraints, weak coulombic or dipolar interactions between penetrating ions and structural elements of the leak determine permselectivity.     

The nature of the conductance increase induced by filipin in cholesterol-containing planar lipid bilayers

The effects of the polyene antibiotic filipin on the conductance and permeability of planar lipid bilayers were investigated under voltage-clamp conditions. The membrane conductance of lipid bilayers containing no cholesterol was not affected by filipin. In the presence of cholesterol containing lipid bilayers, filipin induced a 10(4)-10(5)-fold increase in transmembrane conductance. This conductance increase was dependent on the ionic species present in solution, decreasing in the following order: GCsCl greater than GNaAc greater than GKCl greater than GNaCl greater than CaCl2 greater than GNa2SO4 greater than GBaCl2 greater than GMgCl2. Reversal potential measurements in simple biionic conditions revealed the following relative permeability sequence: PK greater than PCl greater than PNa approximately Pac approximately PBa greater than PCs greater than PMg approximately PCa greater than Psulphate. The filipin-sterol mediated increase in membrane conductance was independent of the membrane potential. The increase in membrane current following a step alteration in membrane potential occurred instantaneously and had no dependence on the previous value of the holding membrane potential. We propose that the filipin-sterol complex forms ion channels in lipid membranes. These channels are found in a single configuration (open state) and select preferentially monovalent cations or anions over divalent ions. Our experimental results are discussed in relation to the effects of other polyene antibiotics on the membrane permeability, and also in relation to experimental problems previously reported with the use of filipin in planar lipid bilayers.