Thermal membrane potential through charged membranes in electrolyte solutions.

Measurements of the thermal membrane potential across cation and anion exchange membranes were carried out by using the same solution of various 1-1 electrolytes on both sides of the membrane. In all cases a good linear relationship was observed between the thermal membrane potential increment psi and the temperature difference increment T. The slope of the linear plot varied with the concentration of the electrolyte. The value of increment psi/increment T versus logarithmic activity of the electrolyte plot was linear with a slope of +/- R/F if the transport number of counterion was unity. The magnitude of increment psi/increment T was independent of coion species but dependent on counterions. These experimental results are in agreement with a theory presented previously. The thermal membrane potential caused by the direct effect of temperature differences and that by the indirect effect arising from the changes in ionic and water chemical potentials due to the temperature difference are separately discussed.     

Thermal membrane potential across charged membranes in NaCl-NH4Cl and LiCl-NH4Cl solutions

Measurements of the thermal membrane potential across cation exchange membranes were carried out by using aqueous solutions containing two 1-1 electrolytes, with an anion in common. The same solution was used on both sides of the membrane. In all cases a good linear relationship was observed between the thermal membrane potential Δψ and the temperature difference ΔT (in the range ΔT = ± 10°C). Assuming that the activity of one cation is equal to that of another cation in the solutions and the sum of transport numbers of cations is unity, the plot of Δψ/ΔT vs logarithmic activity of one cation is linear with a slope of R/F. These experimental results aie in agreement with a theory presented previously. From the analysis of thermal membrane potential in mixtures of electrolytes it is obtained that the cross coefficient of cation-cation interaction in membranes is negative and about 6 to 9% of the main coefficient.     

Sulphate-ion/sodium-ion co-transport by brush-border membrane vesicles isolated from rat kidney cortex

Uptake of SO(4) (2-) into brush-border membrane vesicles isolated from rat kindey cortex by a Ca(2+)-precipitation method was investigated by using a rapid-filtration technique. Uptake of SO(4) (2-) by the vesicles was osmotically sensitive and represented transport into an intra-vesicular space. Transport of SO(4) (2-) by brush-border membranes was stimulated in the presence of Na(+), compared with the presence of K(+) or other univalent cations. A typical ;overshoot' phenomenon was observed in the presence of an NaCl gradient (100mm-Na(+) outside/zero mm-Na(+) inside). Radioactive-SO(4) (2-) exchange was faster in the presence of Na(+) than in the presence of K(+). Addition of gramicidin-D, an ionophore for univalent cations, decreased the Na(+)-gradient-driven SO(4) (2-) uptake. SO(4) (2-) uptake was only saturable in the presence of Na(+). Counter-transport of Na(+)-dependent SO(4) (2-) transport was shown with MoO(4) (2-) and S(2)O(3) (2-), but not with PO(4) (2-). Changing the electrical potential difference across the vesicle membrane by establishing different diffusion potentials (anion replacement; K(+) gradient+/-valinomycin) was not able to alter Na(+)-dependent SO(4) (2-) uptake. The experiments indicate the presence of an electroneutral Na(+)/SO(4) (2-)-co-transport system in brush-border membrane vesicles isolated from rat kidney cortex.     

A sensitive technique for the determination of anion exchange activities in brush-border membrane vesicles. Evidence for two exchangers with different affinities for HCO3- and SITS in rat intestinal epithelium

A large percentage (up to 70%) of 36Cl- influx in brush-border membrane vesicles from rat small intestine under equilibrium exchange conditions was found to be mediated by SITS-inhibitable anion exchange. This Cl-/anion exchange could be measured 10-15-times more sensitive by determining the uptake of trace amounts of 125I- driven by a large Cl- gradient (in greater than out) generated by passing the vesicles through an anion-exchange column. Voltage clamping of the vesicle membrane with K+ and valinomycin did not effect the chloride driven 125I- uptake, showing that the 'overshooting' I- uptake was not mediated by an electrical diffusion potential, as might be generated by the Cl- gradient in the presence of a chloride channel. The Cl-/anion exchange was further characterized in brush-border membrane vesicles from both rat ileum and jejunum by studying the inhibitory action of various anions on the Cl- driven I- uptake. NO3-, Cl-, SCN- and formate at 2 mM could inhibit Cl-/I- exchange for more than 80%. The ileal brush-border membrane vesicles displayed a clear heterogeneity with respect to the inhibitory action of SO2-(4), SITS and HCO-3 on Cl-/I- exchange. Approximately 30% of the Cl-/I- exchange was insensitive to SO2-(4) and showed a relatively low sensitivity to SITS (IC50 = 1 mM) but could be inhibited for 80% by 2 mM HCO-3. Presumably this component represents Cl-/OH- or Cl-/HCO-3 exchange. The residual 70% showed a high sensitivity to SO2-(4) (IC50 = 0.5 mM) and SITS (IC50 = 2.5 microM) but was less sensitive to HCO-3. This part of the exchange activity showed inhibition characteristics very similar to the Cl-/I- exchange in the jejunal vesicles. The latter process was also inhibited for 80% by 2 mM oxalate. As discussed in this paper both exchangers may be involved in the electroneutral transport of NaCl across the apical membrane of the small intestinal villus cell.     

PAT-1 (Slc26a6) is the predominant apical membrane Cl-/HCO3- exchanger in the upper villous epithelium of the murine duodenum

Basal HCO(3)(-) secretion across the duodenum has been shown in several species to principally involve the activity of apical membrane Cl(-)/HCO(3)(-) exchanger(s). To investigate the identity of relevant anion exchanger(s), experiments were performed using wild-type (WT) mice and mice with gene-targeted deletion of the following Cl(-)/HCO(3)(-) exchangers localized to the apical membrane of murine duodenal villi: Slc26a3 [down-regulated in adenoma (DRA)], Slc26a6 [putative anion transporter 1 (PAT-1)], and Slc4a9 [anion exchanger 4 (AE4)]. RT-PCR of the isolated villous epithelium demonstrated PAT-1, DRA, and AE4 mRNA expression. Using the pH-sensitive dye BCECF, anion exchange rates were measured across the apical membrane of epithelial cells in the upper villus of the intact duodenal mucosa. Under basal conditions, Cl(-)/HCO(3)(-) exchange activity was reduced by 65-80% in the PAT-1(-) duodenum, 30-40% in the DRA(-) duodenum, and <5% in the AE4(-) duodenum compared with the WT duodenum. SO(4)(2-)/HCO(3)(-) exchange was eliminated in the PAT-1(-) duodenum but was not affected in the DRA(-) and AE4(-) duodenum relative to the WT duodenum. Intracellular pH (pH(i)) was reduced in the PAT-1(-) villous epithelium but increased to WT levels in the absence of CO(2)/HCO(3)(-) or during methazolamide treatment. Further experiments under physiological conditions indicated active pH(i) compensation in the PAT-1(-) villous epithelium by combined activities of Na(+)/H(+) exchanger 1 and Cl(-)-dependent transport processes at the basolateral membrane. We conclude that 1) PAT-1 is the major contributor to basal Cl(-)/HCO(3)(-) and SO(4)(2-)/HCO(3)(-) exchange across the apical membrane and 2) PAT-1 plays a role in pH(i) regulation in the upper villous epithelium of the murine duodenum.     

Mechanism of energization of uptake of the fluorescent dye 2-(4-dimethylaminostyryl)-1-ethylpyridinium cation [DMP+] into an acrA strain of Escherichia coli.

The mechanism of uptake of the fluorescent dye 2-(4-dimethylaminostyryl)-1-ethylpyridinium cation (DMP+) into cells and vesicles of the acrA strain AS-1 of Escherichia coli was examined. Uptake was energized by substrate oxidation and discharged by uncouplers. Uptake was enhanced by the presence of tetraphenylphosphonium cation, tetraphenylboron anion and tributyltin chloride, which may inhibit the efflux system for DMP+. Uptake was inhibited by 5-methoxyindole-2-carboxylic acid (MIC). By the use of ionophores with right-side-out vesicles loaded with monovalent cations it was shown that DMP+ uptake could be driven both by the establishment of a membrane potential across the vesicle membrane and by a H+/DMP+ antiport system. Attempts to demonstrate the latter mechanism in everted membrane vesicles were unsuccessful.     

Mouse Ae1 E699Q mediates SO42-i/anion-o exchange with [SO42-]i-dependent reversal of wild-type pHo sensitivity

The SLC4A1/AE1 gene encodes the electroneutral Cl(-)/HCO(3)(-) exchanger of erythrocytes and renal type A intercalated cells. AE1 mutations cause familial spherocytic and stomatocytic anemias, ovalocytosis, and distal renal tubular acidosis. The mutant mouse Ae1 polypeptide E699Q expressed in Xenopus oocytes cannot mediate Cl(-)/HCO(3)(-) exchange or (36)Cl(-) efflux but exhibits enhanced dual sulfate efflux mechanisms: electroneutral exchange of intracellular sulfate for extracellular sulfate (SO(4)(2-)(i)/SO(4)(2-)(o) exchange), and electrogenic exchange of intracellular sulfate for extracellular chloride (SO(4)(2-)(i)/Cl(-)(o) exchange). Whereas wild-type AE1 mediates 1:1 H(+)/SO(4)(2-) cotransport in exchange for either Cl(-) or for the H(+)/SO(4)(2-) ion pair, mutant Ae1 E699Q transports sulfate without cotransport of protons, similar to human erythrocyte AE1 in which the corresponding E681 carboxylate has been chemically converted to the alcohol (hAE1 E681OH). We now show that in contrast to the normal cis-stimulation by protons of wild-type AE1-mediated SO(4)(2-) transport, both SO(4)(2-)(i)/Cl(-)(o) exchange and SO(4)(2-)(i)/SO(4)(2-)(o) exchange mediated by mutant Ae1 E699Q are inhibited by acidic pH(o) and activated by alkaline pH(o). hAE1 E681OH displays a similarly altered pH(o) dependence of SO(4)(2-)(i)/Cl(-)(o) exchange. Elevated [SO(4)(2-)](i) increases the K(1/2) of Ae1 E699Q for both extracellular Cl(-) and SO(4)(2-), while reducing inhibition of both exchange mechanisms by acid pH(o). The E699Q mutation also leads to increased potency of self-inhibition by extracellular SO(4)(2-). Study of the Ae1 E699Q mutation has revealed the existence of a novel pH-regulatory site of the Ae1 polypeptide and should continue to provide valuable paths toward understanding substrate selectivity and self-inhibition in SLC4 anion transporters.     

Role of tubular secretion and carbonic anhydrase in vertebrate renal sulfate excretion

The renal proximal tubule of vertebrates performs an essential role in controlling plasma SO(4)(2-) concentration ([SO(4)(2-)]). Although net tubular SO(4)(2-) reabsorption is the predominate control process in terrestrial vertebrates, a facilitated secretory flux is also present. In contrast, marine teleosts obtain excess SO(4)(2-) from drinking, and increased plasma [SO(4)(2-)] is prevented predominately through net tubular secretion. Tubular SO(4)(2-) secretion is accomplished by at least two electroneutral anion exchange processes in series. Movement of SO(4)(2-) into the cell across the basolateral membrane is pH dependent, suggesting SO(4)(2-)/OH(-) exchange. Luminal HCO(3)(-) and Cl(-) can facilitate SO(4)(2-) movement out of the cell across the brush-border membrane. The molecular identities of the anion exchangers are unknown but are probably homologues of SO(4)(2-) transporters in the mammalian SLC26 gene family. In all species tested, glucocorticoids increase renal SO(4)(2-) excretion. Whereas glucocorticoids downregulate SO(4)(2-) reabsorptive mechanisms in terrestrial vertebrates, they may also stimulate a mediated secretory flux. In the marine teleost, cortisol increases the level of SO(4)(2-)/HCO(3)(-) exchange at the brush-border membrane, tubular carbonic anhydrase (CA) activity, CAII protein, and a proportion of tubular SO(4)(2-) secretion that is CA dependent. CA activity is required for about one-half of this net SO(4)(2-) secretion but is also required for about one-half of the net reabsorption in bird proximal epithelium. A CA-SO(4)(2-)/anion exchanger metabolon arrangement is proposed that may speed both the secretory and reabsorptive processes.     

Proton pathways in rat renal brush-border and basolateral membranes

The quenching of acridine orange fluorescence was used to monitor the formation and dissipation of pH gradients in brush-border and basolateral membrane vesicles isolated from rat kidney cortex. The fluorescence changes of acridine orange were shown to be sensitive exclusively to transmembrane delta pH and not to membrane potential difference. In brush-border membrane vesicles, an Na+ (Li+)-H+ exchange was confirmed. At physiological Na+ concentrations, 40-70% of Na+-H+ exchange was mediated by the electroneutral Na+-H+ antiporter; the remainder consisted of Na+ and H+ movements through parallel conductive pathways. Both modes of Na+-H+ exchange were saturable, with half-maximal rates at about 13 and 24 mM Na+, respectively. Besides a Na+ gradient, a K+ gradient was also able to produce an intravesicular acidification, demonstrating conductance pathways for H+ and K+ in brush-border membranes. Experiments with Cl- or SO2-4 gradients failed to demonstrate measurable Cl--OH- or SO2-4-OH- exchange by an electroneutral antiporter in brush-border membrane vesicles; only Cl- conductance was found. In basolateral membrane vesicles, neither Na+(Li+)-H+ exchange nor Na+ or K+ conductances were found. However, in the presence of valinomycin-induced K+ diffusion potential, H+ conductance of basolateral membranes was demonstrated, which was unaffected by ethoxzolamide and 4,4'-diisothiocyanostilbene-2,2-disulfonic acid. A Cl- conductance of the membranes was also found, but antiporter-mediated electroneutral Cl--OH- or SO2-4-OH- exchange could not be detected by the dye method. The restriction of the electroneutral Na+-H+ exchanger to the luminal membrane can explain net secretion of protons in the mammalian proximal tubule which leads to the reabsorption of bicarbonate.     

Rapid electrogenic sulfate-chloride exchange mediated by chemically modified band 3 in human erythrocytes

One of the modes of action of the red blood cell anion transport protein is the electrically silent net exchange of 1 Cl- for 1 SO4= and 1 H+. Net SO4(=)-Cl- exchange is accelerated by low pH or by conversion of the side chain of glutamate 681 into an alcohol by treatment of intact cells with Woodward's reagent K (WRK) and BH4-. The studies described here were performed to characterize the electrical properties of net SO4(=)-Cl- exchange in cells modified with WRK/BH4-. The SO4= conductance measured in 100 mM SO4= medium is smaller in modified cells than in control cells. However, the efflux of [35S] SO4= into a 150-mM KCl medium is 80-fold larger in modified cells than in control cells and is inhibited 99% by 10 microM H2DIDS. No detectable H+ flux is associated with SO4(=)-Cl- exchange in modified cells. In the presence of gramicidin to increase the cation permeability, the stoichiometry of SO4(=)-Cl- exchange is not distinguishable from 1:1. In modified cells loaded with SO4=, the valinomycin-mediated efflux of 86Rb+ into an Na- gluconate medium is immediately stimulated by the addition of 5 mM extracellular Cl-. Therefore, SO4(=)-Cl- exchange in modified cells causes an outward movement of negative charge, as expected for an obligatory 1:1 SO4(=)-Cl- exchange. This is the first example of an obligatory, electrogenic exchange process in band 3 and demonstrates that the coupling between influx and efflux does not require that the overall exchange be electrically neutral. The effects of membrane potential on SO4(=)-SO4= exchange and SO4(=)-Cl- exchange in modified cells are consistent with a model in which nearly a full net positive charge moves inward through the transmembrane field during the inward Cl- translocation event, and a small net negative charge moves with SO4= during the SO4= translocation event. This result suggests that, in normal cells, the negative charge on Glu 681 traverses most of the transmembrane electric field, accompanied by Cl- and the equivalent of two protein-bound positive charges.     

Effect of phloretin on the permeability of thin lipid membranes

Phloretin dramatically increases cation conductances and decreases anion conductances of membranes treated with ion carriers (nonactin, valinomycin, carbonyl-cyanide-m-chlorophenylhydrazone [CCCP], and Hg(C6F5)2) or lipophilic ions (tetraphenylarsonium [tphAs+] and tetraphenylborate [TPhB-]). For example, on phosphatidylethanolamine membranes, 10(-4) M phloretin increases K+ -nonactin and TPhAs+ conductances and decreases CCCP- and TPhB- conductances 10(3)-fold; on lecithin: cholesterol membranes, it increases K+-nonactin conductance 10(5)-fold and decreases CCCP- conductance 10(3)-fold. Similar effects are obtained with p- and m-nitrophenol at 10(-2) M. These effects are produced by the un-ionized form of phloretin and the nitrophenols. We believe that phloretin, which possesses a large dipole moment, adsorbs and orients at the membrane surface to introduce a dipole potential of opposite polarity to the preexisting positive one, thus increasing the partition coefficient of cations into the membrane interior and decreasing the partition coefficient of anions. (Phloretin may also increase the fluidity of cholesterol-containing membranes; this is manifested by its two- to three-fold increase in nonelectrolyte permeability and its asymmetrical effect on cation and anion conductances in cholesterol-containing membranes.) It is possible that pholoretin's inhibition of chloride, urea, and glucose transport in biological membranes results from the effects of these intense intrafacial dipole fields on the translocator(s) of these molecules.     

Enhanced formation of a HCO3- transport metabolon in exocrine cells of Nhe1-/- mice

Cl(-) influx across the basolateral membrane is a limiting step in fluid production in exocrine cells and often involves functionally linked Cl(-)/HCO(3)(-) (Ae) and Na(+)/H(+) (Nhe) exchange mechanisms. The dependence of this major Cl(-) uptake pathway on Na(+)/H(+) exchanger expression was examined in the parotid acinar cells of Nhe1(-/-) and Nhe2(-/-) mice, both of which exhibited impaired fluid secretion. No change in Cl(-)/HCO(3)(-) exchanger activity was detected in Nhe2-deficient mice. Conversely, Cl(-)/HCO(3)(-) exchanger activity increased nearly 4-fold in Nhe1-deficient mice, despite only minimal or any change in mRNA and protein levels of the anion exchanger Ae2. Acetazolamide completely blocked the increase in Cl(-)/HCO(3)(-) exchanger activity in Nhe1-null mice suggesting that increased anion exchange required carbonic anhydrase activity. Indeed, the parotid glands of Nhe1(-/-) mice expressed higher levels of carbonic anhydrase 2 (Car2) polypeptide. Moreover, the enhanced Cl(-)/HCO(3)(-) exchange activity was accompanied by an increased abundance of Car2.Ae2 complexes in the parotid plasma membranes of Nhe1(-/-) mice. Anion exchanger activity was also significantly reduced in Car2-deficient mice, consistent with an important role of a putative Car2.Ae2 HCO(3)(-) transport metabolon in parotid exocrine cell function. Increased abundance of this HCO(3)(-) transport metabolon is likely one of the multiple compensatory changes in the exocrine parotid gland of Nhe1(-/-) mice that together attenuate the severity of in vivo electrolyte and acid-base balance perturbations.     

Chloride conductance of CFTR facilitates basal Cl-/HCO3- exchange in the villous epithelium of intact murine duodenum

Villi of the proximal duodenum are situated for direct exposure to gastric acid chyme. However, little is known about active bicarbonate secretion across villi that maintains the protective alkaline mucus barrier, a process that may be compromised in cystic fibrosis (CF), i.e., in the absence of a functional CF transmembrane conductance regulator (CFTR) anion channel. We investigated Cl(-)/HCO(3)(-) exchange activity across the apical membrane of epithelial cells located at the midregion of villi in intact duodenal mucosa from wild-type (WT) and CF mice using the pH-sensitive dye BCECF. Under basal conditions, the Cl(-)/HCO(3)(-) exchange rate was reduced by approximately 35% in CF compared with WT villous epithelium. Cl(-)/HCO(3)(-) exchange in WT and CF villi responded similarly to inhibitors of anion exchange, and membrane depolarization enhanced rates of Cl(-)(out)/HCO(3)(-)(in) exchange in both epithelia. In anion substitution studies, anion(in)/HCO(3)(-)(out) exchange rates were greater in WT epithelium using Cl(-) or NO(3)(-), but decreased to the level of the CF epithelium using the CFTR-impermeant anion, SO(4)(2-). Similarly, treatment of WT epithelium with the CFTR-selective blocker glybenclamide decreased the Cl(-)/HCO(3)(-) exchange rate to the level of CF epithelium. The mRNA expression of Slc26a3 (downregulated in adenoma) and Slc26a6 (putative anion exchanger-1) was similar between WT and CF duodena. From these studies of murine duodenum, we conclude 1) characteristics of Cl(-)/HCO(3)(-) exchange in the villous epithelium are most consistent with Slc26a6 activity, and 2) Cl(-) channel activity of CFTR facilitates apical membrane Cl(-)(in)/HCO(3)(-)(out) exchange by providing a Cl(-) "leak" under basal conditions.     

Native and recombinant Slc26a3 (downregulated in adenoma, Dra) do not exhibit properties of 2Cl-/1HCO3- exchange

The recent proposal that Dra/Slc26a3 mediates electrogenic 2Cl(-)/1HCO(3)(-) exchange suggests a required revision of classical concepts of electroneutral Cl(-) transport across epithelia such as the intestine. We investigated 1) the effect of endogenous Dra Cl(-)/HCO(3)(-) activity on apical membrane potential (V(a)) of the cecal surface epithelium using wild-type (WT) and knockout (KO) mice; and 2) the electrical properties of Cl(-)/(OH(-))HCO(3)(-) exchange by mouse and human orthologs of Dra expressed in Xenopus oocytes. Ex vivo (36)Cl(-) fluxes and microfluorometry revealed that cecal Cl(-)/HCO(3)(-) exchange was abolished in the Dra KO without concordant changes in short-circuit current. In microelectrode studies, baseline V(a) of Dra KO surface epithelium was slightly hyperpolarized relative to WT but depolarized to the same extent as WT during luminal Cl(-) substitution. Subsequent studies indicated that Cl(-)-dependent V(a) depolarization requires the anion channel Cftr. Oocyte studies demonstrated that Dra-mediated exchange of intracellular Cl(-) for extracellular HCO(3)(-) is accompanied by slow hyperpolarization and a modest outward current, but that the steady-state current-voltage relationship is unaffected by Cl(-) removal or pharmacological blockade. Further, Dra-dependent (36)Cl(-) efflux was voltage-insensitive in oocytes coexpressing the cation channels ENaC or ROMK. We conclude that 1) endogenous Dra and recombinant human/mouse Dra orthologs do not exhibit electrogenic 2Cl(-)/1HCO(3)(-) exchange; and 2) acute induction of Dra Cl(-)/HCO(3)(-) exchange is associated with secondary membrane potential changes representing homeostatic responses. Thus, participation of Dra in coupled NaCl absorption and in uncoupled HCO(3)(-) secretion remains compatible with electroneutrality of these processes, and with the utility of electroneutral transport models for predicting epithelial responses in health and disease.     

Relationship among the surface potential, Donnan potential and charge density of ion-penetrable membranes

Calculation of the potential distribution across a uniformly charged ion-penetrable membrane that we developed previously is extended to derive a relationship among the surface potential, Donnan potential and charge density of an ion-penetrable membrane in a mixed solution of 2-1 and 1-1 electrolytes. We also present an approximate method for calculating the surface potential/Donnan potential/charge density relationship for membranes with an arbitrary distribution of membrane-fixed charges.     

INFLUENCE OF A SLIGHT MODIFICATION OF THE COLLODION MEMBRANE ON THE SIGN OF THE ELECTRIFICATION OF WATER

1. It is shown that collodion membranes which have received one treatment with a 1 per cent gelatin solution show for a long time (if not permanently) afterwards a different osmotic behavior from collodion membranes not treated with gelatin. This difference shows itself only towards solutions of those electrolytes which have a tendency to induce a negative electrification of the water particles diffusing through the membrane, namely solutions of acids, acid salts, and of salts with trivalent and tetravalent cations; while the osmotic behavior of the two types of membranes towards solutions of salts and alkalies, which induce a positive electrification of the water particles diffusing through the membrane, is the same. 2. When we separate solutions of salts with trivalent cation, e.g. LaCl₃ or AlCl₃, from pure water by a collodion membrane treated with gelatin, water diffuses rapidly into the solution; while no water diffuses into the solution when the collodion membrane has received no gelatin treatment. 3. When we separate solutions of acid from pure water by a membrane previously treated with gelatin, negative osmosis occurs; i.e., practically no water can diffuse into the solution, while the molecules of solution and some water diffuse out. When we separate solutions of acid from pure water by collodion membranes not treated with gelatin, positive osmosis will occur; i.e., water will diffuse rapidly into the solution and the more rapidly the higher the valency of the anion. 4. These differences occur only in that range of concentrations of electrolytes inside of which the forces determining the rate of diffusion of water through the membrane are predominantly electrical; i.e., in concentrations from 0 to about M/16. For higher concentrations of the same electrolytes, where the forces determining the rate of diffusion are molecular, the osmotic behavior of the two types of membranes is essentially the same. 5. The differences in the osmotic behavior of the two types of membranes are not due to differences in the permeability of the membranes for solutes since it is shown that acids diffuse with the same rate through both kinds of membranes. 6. It is shown that the differences in the osmotic behavior of the two types of collodion membranes towards solutions of acids and of salts with trivalent cation are due to the fact that in the presence of these electrolytes water diffuses in the form of negatively charged particles through the membranes previously treated with gelatin, and in the form of positively charged particles through collodion membranes not treated with gelatin. 7. A treatment of the collodion membranes with casein, egg albumin, blood albumin, or edestin affects the behavior of the membrane towards salts with trivalent or tetravalent cations and towards acids in the same way as does a treatment with gelatin; while a treatment of the membranes with peptone prepared from egg albumin, with alanine, or with starch has no such effect.     

Aged human erythrocytes exhibit increased anion exchange

Young and old erythrocytes show different rate constants of anion exchange as measured by 35SO4(2-) efflux at 37 degrees C. Results indicate that the rate constant for 35SO4(2-) efflux (SO2-4-Cl- exchange) from old cells is approximately 20% greater than from young less dense cells. The cell water volume of older cells is also decreased. Based on these results and previously reported decreases of cell membrane area in aged cells we conclude that anion exchange (35SO4(2-)) is increased in older, more dense human erythrocytes.     

The effects of dansylation on the pH dependence of SO4(2-) and Cl- equilibrium exchange and on the H+/SO4(2-) cotransport across the red blood cell membrane

Treatment of the erythrocyte membrane with dansyl chloride leads to the following effects: (i) SO4(2-) transport is enhanced, Cl- transport is reduced. At maximal acceleration of sulfate exchange, Cl- exchange is only partially inhibited. The two effects are lineary related suggesting that the Cl- and SO4(2-) transporting forms of band 3 are derived from the same pool. (ii) The maximum of the pH dependence of SO4(2-) equilibrium exchange as measured at low sulfate concentrations is replaced by a plateau. It now resembles the pH dependence of Cl- exchange in untreated red cells. The pH dependence of SO4(2-) equilibrium exchange as measured at high sulfate concentrations is virtually unchanged after dansylation. The pH dependence of the partially inhibited Cl- equilibrium exchange across the dansylated membrane as measured at high chloride concentrations remains similar as in the untreated red cells but is somewhat less pronounced. (iii) SO4(2-)/H+ cotransport remains essentially unaltered after modification by dansyl chloride. The effects of dansylation are discussed in terms of a model similar to the titratable carrier model originally proposed by Gunn (Gunn, R.B. (1972) in Oxygen Affinity of Hemoglobin and Red Cell Acid Base Status (Rorth, M. and Astrup, P., eds.), pp. 823-827, Munksgaard, Copenhagen).     

The hydrogen ion-pumping adenosine triphosphatase of platelet dense granule membrane. Differences from F1F0- and phosphoenzyme-type ATPases

Using a coupled transport assay which detects only those ATPase molecules functionally inserted into the platelet dense granule membrane, we have characterized the inhibitor sensitivity, substrate specificity, and divalent cation requirements of the granule H+ pump. Under identical assay conditions, the granule ATPase was insensitive to concentrations of NaN3, oligomycin, and efrapeptin which almost completely inhibit ATP hydrolysis by mitochondrial membranes. The granule ATPase was inhibited by dicyclohexylcarbodiimide but only at concentrations much higher than those needed to maximally inhibit mitochondrial ATPase. Vanadate (VO3-) ion and ouabain also failed to inhibit granule ATPase activity at concentrations which maximally inhibited purified Na+,K+-ATPase. Two alkylating agents, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole and N-ethylmaleimide both completely inhibited H+ pumping by the granule ATPase under conditions where ATP hydrolysis by mitochondrial membranes or Na+,K+-ATPase was hardly affected. These results suggest that the H+-pumping ATPase of platelet granule membrane may belong to a class of ion-translocating ATPases distinct from both the phosphoenzyme-type ATPases present in plasma membrane and the F1F0-ATPases of energy-transducing membranes.