Transmembrane Electrical Potential Difference Research Articles

Kinetics of ATP synthesis catalyzed by the H(+)-ATPase from chloroplasts (CF0F1) reconstituted into liposomes and coreconstituted with bacteriorhodopsin.

The H(+)-ATPase from chloroplasts (CF0F1) was isolated, purified and reconstituted into liposomes from phosphatidylcholine/phosphatidic acid. A transmembrane pH difference, delta pH, and a transmembrane electric potential difference, delta psi, were generated by an acid/base transition. The rate of ATP synthesis was measured at constant delta pH and constant delta psi as a function of temperature between 5 degrees C and 45 degrees C. The activation energy was 55 kJ mol-1. CF0F1 was coreconstituted with bacteriorhodopsin at a molar ratio of approximately 1:170 in the same type of liposomes. Illumination of the proteoliposomes leads to proton transport into the vesicles generating a constant delta pH = 1.8. The dependence of the rate of ATP synthesis on ADP concentration was measured with CF0F1 in the oxidized state, E(ox), and in the reduced state, E(red). The results can be described by Michaelis-Menten kinetics with the following parameters: Vmax = 0.5 s-1, Km = 8 microM for E(ox) and Vmax = 2.0 s-1, Km = 8 microM for E(red).

Abscisic acid effects on intracellular pH and ion fluxes in barley leaf segments

Changes in intracellular pH and in H+, K+ and Cl− fluxes were evaluated in different experimental conditions in leaf segments of barley (Hordeum vulgare cv. Georgie) incubated in the dark, at pH 5.5, in the presence or absence of abscisic acid (ABA), and a comparison was made between the effects of ABA and those of erythrosin B (EB), a plasmalemma H+‐pump inhibitor. In all conditions tested, ABA induced a cell sap acidification, an alkalinization of the external medium, a decrease in K+ intracellular contents, and an increase in the contents of Cl−. The ABA‐induced decrease in K+ content was chiefly due to the inhibition of K+ influx. On the contrary, ABA did not influence the uptake of Cl−, but inhibited Cl− efflux, the inhibition satisfactorily accounting for the larger Cl− content observed in the presence of the hormone. The intracellular acidification and the decrease in apparent outward net transport of H+ observed with ABA were seemingly not associated with the activity of the proton pump, the transmembrane electrical potential difference, or K+ transport. On the contrary, a correlation was evident with the changes in Cl− content. These results and, in particular, the similarity between the effects of ABA and those induced by 4,4 ‐diisothiocyano‐2,2‐disulfonic acid stilbene (DIDS), a Cl− channel‐blocking agent, suggest that the ABA‐induced changes in intracellular pH and in H+ transport might depend on the capability of ABA to inhibit Cl− efflux, more than on a primary inhibition of the H+ pump, and propose an important role for ABA in regulating the Cl− channels.

Studies of the Uptake of Nitrate in Barley

Transmembrane electrical potential differences (Deltapsi) of epidermal and cortical cells were measured in intact roots of barley (Hordeum vulgare L. cv Klondike). The effects of exogenous NO(3) (-) on Deltapsi (in the concentration range from 100 micromolar to 20 millimolar) were investigated to probe the mechanisms of nitrate uptake by the high-affinity (HATS) and low-affinity (LATS) transport systems for NO(3) (-) uptake. Both transport systems caused depolarization of Deltapsi, demonstrating that the LATS (like the HATS) for NO(3) (-) uptake is probably mediated by an electrogenic cation (H(+)?) cotransport system. Membrane depolarization by the HATS was "inducible" by NO(3) (-), and saturable with respect to exogenous [NO(3) (-)]. By contrast, depolarization by the LATS was constitutive, and first-order in response to external [NO(3) (-)]. H(+) fluxes, measured in 200 micromolar and in 5 millimolar Ca(NO(3))(2) solutions, failed to alkalinize external media as anticipated for a 2 H(+):1 NO(3) (-) symport. However, switching from K(2)SO(4) solutions (which were strongly acidifying) to KNO(3) solutions at the same K(+) concentration caused marked reductions in H(+) efflux. These observations are consistent with NO(3) (-) uptake by the HATS and the LATS via 2 H(+):1 NO(3) (-) symports. These observations establish that the HATS for nitrate uptake by barley roots is essentially similar to those reported for Lemna and Zea mays by earlier workers. There are, nevertheless, distinct differences between barley and corn in their quantitative responses to external NO(3) (-).

Transmembrane electrical potential difference regulates Na+/HCO3- cotransport and intracellular pH in hepatocytes.

We have examined the hypothesis that a regulatory interplay between pH-regulated plasma membrane K+ conductance (gK+) and electrogenic Na+/HCO3- cotransport contributes importantly to regulation of intracellular pH (pHi) in hepatocytes. In individual cells, membrane depolarization produced by transient exposure to 50 mM K+ caused a reversible increase in pHi in the presence, but not absence, of HCO3-, consistent with voltage-dependent HCO3- influx. In the absence of HCO3-, intracellular alkalinization and acidification produced by NH4Cl exposure and withdrawal produced membrane hyperpolarization and depolarization, respectively, as expected for pHi-induced changes in gK+. By contrast, in the presence of HCO3-, NH4Cl exposure and withdrawal produced a decrease in apparent buffering capacity and changes in membrane potential difference consistent with compensatory regulation of electrogenic Na+/HCO3- cotransport. Moreover, the rate of pHi and potential difference recovery was several-fold greater in the presence as compared with the absence of HCO3-. Finally, continuous exposure to 10% CO2 in the presence of HCO3- produced intracellular acidification, and the rate of pHi recovery from intracellular acidosis was inhibited by Ba2+, which blocks pHi-induced changes in gK+, and by 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid, which inhibits Na+/HCO3- cotransport. These findings suggest that in hepatocytes, changes in transmembrane electrical potential difference, mediated by pH-sensitive gK+, play a central role in regulation of pHi through effects on electrogenic Na+/HCO3- cotransport.

The K+-Driven Amino Acid Cotransporter of the Larval Midgut of Lepidoptera: Is Na+ an Alternative Substrate?

ABSTRACT Amino acid accumulation within brush-border membrane vesicles (BBMV) from the larval midgut of Lepidoptera is driven by a K+ gradient. However, it can also be driven by a Na+ gradient, although with reduced efficiency. To examine the possibility that sodium and potassium ions are handled by the same amino acid transporter, glycine uptake into BBMV from Philosamia cynthia Drury was measured in the presence of a pH gradient and of a transmembrane electrical potential difference, i.e. in simulated ‘physiological’ conditions. The kinetics of glycine uptake at extravesicular saturating Na+ or K+ concentrations discloses a higher affinity of the cotransporter for the amino acid in the presence of Na+ but a maximum transport rate with K+. Glycine uptake at a fixed concentration as a function of external Na+ or K+ concentration yields curves that show saturation but do not fit a rectangular hyperbola, with Hill coefficients less than 1 with Na+ and greater than 1 with K+. These coefficients vary according to glycine concentration. Increasing the concentration of extravesicular Na+ at a saturating external K+ concentration reduced glycine uptake to 70% of the control value. This inhibition curve is compatible with competition between the two cations for the same cotransporter and with the presence of different kinetic constants with Na+ or K+. The data are consistent with a steady-state random two-substrate mechanism for glycine transport, with Na+ and K+ as alternative substrates.

Early Effects of Penconazole on H+ and K+ Transport, Electrolyte Leakage and Transmembrane Electrical Potential in Egeria densa Leaves

AbstractThe early effects of penconazole (PCZ) at relatively high concentration (10−4 to 5 × 10−4 M) on changes in pH and in titratable acidity of the medium, transmembrane electrical potential difference (Em), electrolyte leakage and cell morphology were investigated in Egeria densa leaves. At the lowest (10−4 M) concentration and in the presence of a very low (10 μM) K+ concentration, triazole induced an early, moderate hyperpolarization of Em, associated with a decrease of net K+ uptake, suggesting some increase in the passive permeability to K+. This Em hyperpolarization was no longer detectable at high (2 mM) K+out concentration. At high PCZ concentrations (3 × 10−4 M and 5 × 10−4 M) the early hyperpolarization detectable in the presence of a low K+out concentration became transient, and was followed by a marked depolarization. PCZ, at these concentrations, suppressed acidification of the medium, stimulated electrolyte leakage and, in the mesophyll cells, induced some shrinking of the cytoplasm and its disconnection from the cell walls. These results are interpreted as due to an early effect of this triazole leading to the disorganization of the plasma membrane.

Mutations Affecting Internal TEA Blockade Identify the Probable Pore-Forming Region of a K + Channel

The active site of voltage-activated potassium channels is a transmembrane aqueous pore that permits ions to permeate the cell membrane in a rapid yet highly selective manner. A useful probe for the pore of potassium-selective channels is the organic ion tetraethylammonium (TEA), which binds with millimolar affinity to the intracellular opening of the pore and blocks potassium current. In the potassium channel encoded by the Drosophila Shaker gene, an amino acid residue that specifically affects the affinity for intracellular TEA has now been identified by site-directed mutagenesis. This residue is in the middle of a conserved stretch of 18 amino acids that separates two locations that are both near the external opening of the pore. These findings suggest that this conserved region is intimately involved in the formation of the ion conduction pore of voltage-activated potassium channels. Further, a stretch of only eight amino acid residues must traverse 80 percent of the transmembrane electric potential difference.

Gramicidin in chromatophores of Rhodobacter sphaeroides

Chromatophores of Rhodobacter sphaeroides were excited with light flashes to generate a transmembrane electrical potential difference. The electric relaxation was measured by electrochromic absorption changes as a function of added gramicidin. At low gramicidin/bacteriochlorophyll (BChl) molar ratios the decay of the electrochromic absorption changes showed a biphasic behaviour, with a fast phase relaxing at some μs, and a slow phase relaxing at more than 100 ms. This was attributable to a mixture of vesicles containing gramicidin dimers with others containing none. The concentration dependence of this effect was linear. This implied full dimerization of gramicidin. The data were interpreted to yield an average bacteriochlorophyll content per chromatophore of 770(±150) and the conductance of a single gramicidin dimer in the chromatophore membrane of 15(±4) pS (in about 115 mM KCl).

Sodium and calcium share the electrogenic 2 Na(+)-1 H+ antiporter in crustacean antennal glands

Na uptake by short-circuited epithelial brush-border membrane vesicles of Atlantic lobster (Homarus americanus) antennal gland labyrinth was Cl independent, amiloride sensitive, and stimulated by a transmembrane H+ gradient [( H]i greater than [H]o; i is internal, o is external). Na influx (2.5-s uptake) was a sigmoidal function of [Na]o (25-400 mM) when pHi = 5.0 and pHo = 8.0 and followed the Hill equation for binding cooperatively [apparent maximal influx (Jmax) = 271 nmol.mg protein-1.s-1, apparent affinity constant for Na (KNa) = 310 mM Na, and Hill coefficient (n) = 2.41]. Amiloride acted as a competitive inhibitor of Na binding to two external sites with markedly dissimilar apparent amiloride affinities (Ki1 = 14 microM; Ki2 = 1,340 mM). Electrogenic Na-H antiport by these vesicles was demonstrated by equilibrium-shift experiments in which an imposed transmembrane electrical potential difference was the only driving force for exchange. A transport stoichiometry of 2 Na to 1 H was demonstrated with the static-head technique in which a balance of driving forces was attained with 10:1 Na gradient and 100:1 H gradient. External Ca, like amiloride, was a strong competitive inhibitor of Na-H exchange, acting at two sites on the outer vesicular face with markedly different apparent divalent cation affinities (Ki1 = 20 microM; Ki2 = 500 microM). Ca-H exchange by electrogenic Na-H antiporter was demonstrated in complete absence of Na by use of an outward H gradient in presence and absence of amiloride. Both external amiloride (Ki1 = 70 microM; Ki2 = 500 microM) and Na (Ki1 = 12 mM; Ki2 = 380 mM) were competitive inhibitors of Ca-H exchange. These results suggest that the electrogenic 2 Na-1 H exchanger characterized for this crustacean epithelium may also have a role in organismic Ca balance.

High Sensitivity to Auxin is a Common Feature of Hairy Root

The responses to auxin of Lycopersicon esculentum roots transformed by (T(l)+T(r))-DNA of the Ri plasmid of agropine-type Agrobacterium rhizogenes strain 15834 and Catharanthus trichophyllus roots transformed by the (T(l)+T(r))-DNA, and by T(l)- or T(r)- DNA alone of the same bacterial strain were compared to that of their normal counterparts. The transmembrane electrical potential difference of root protoplasts was measured as a function of the concentration of exogenous naphthalene acetic acid. The sensitivity to auxin expressed by this response was shown to be independent of the measurement conditions and of the basal polarization of isolated protoplasts. According to this electrical response, as well as to the modulation by auxin of proton excretion by root tips and root tip elongation, roots transformed by (T(l)+T(r)) DNA are 100 to 1000 times more sensitive to exogenous auxin than normal roots, as is the case with normal and transformed roots from Lotus corniculatus (WH Shen, A Petit, J Guern, J Tempé [1988] Proc Natl Acad Sci USA 85: 3417-3421). Further-more, transformed roots of C. trichophyllus are not modified in their sensitivity to fusicoccin, illustrating the specificity of the modification of the auxin sensitivity. Roots transformed by the T(r)-DNA alone showed the same sensitivity to auxin as normal roots, whereas the roots transformed by the T(l)-DNA alone exhibited an auxin sensitivity as high as the roots transformed by (T(l)+T(r))-DNA. It was concluded that the high sensitivity to auxin is controlled by the T(l)-DNA in agropine type Ri plasmids.

Evaluation of the buffer capacity and permeability constant for protons in chromatophores from Rhodobacter capsulatus

1. The kinetics of decay in the dark of the transmembrane pH difference (delta pH) induced by light in nonphosphorylating chromatophores of Rhodobacter capsulatus were studied using the fluorescent probe 9-aminoacridine, in the presence of 50 mM KCl and 2 microM valinomycin. The transient fluorescence changes induced by acid to base transitions of chromatophore suspensions were used as an empirical calibration [Casadio, R. & Melandri, B. A. (1985) Arch. Biophys. Biochem. 238, 219-228]. The kinetic competence of the probe response was tested by accelerating the delta pH decay with the ionophore nigericin. 2. The time course in the dark of the increase in the internal pH in pre-illuminated chromatophores was analyzed on the basis of a model which assumes a certain number of internal buffers in equilibrium with the free protons and a diffusion-controlled H+ efflux [Whitmarsh, J. (1987) Photosynt. Res. 12, 43-62]. This model was extended to include the effects of the transmembrane electric potential difference on the H+ efflux. 3. The diffusion constant for proton efflux was measured at different values of the internal pH by evaluating the frequency of trains of single-turnover flashes capable of maintaining different delta pH in a steady state. The steady-state equation derived from the model does not include any parameter relative to the internal buffers and allows unequivocal determination of the diffusion constant on the basis of the known H+/e- ratio (equal to two) for the active proton translocation by the bacterial photosynthetic chain. A value for the first-order diffusion constant corresponding to a permeability coefficient, PH = 0.2 micron.s-1, was obtained at an external pH of 8.0; this value was constant for an internal pH ranging over 7.0-4.7. 4. Using the value of the diffusion constant determined experimentally, a satisfactory fitting of the kinetics of delta pH decay in the dark could be obtained when the presence of two internal buffers (with pK values of 3.6 and 6.7, respectively) was assumed. For these calculations, the time course of the transmembrane electric potential difference was evaluated from the electrochromic signal of carotenoids, calibrated with K(+)-induced diffusion potentials. The two internal buffers, suitable for modelling the behaviour of the system, were at concentrations of 250 mM (pK = 3.6) and 24 mM (pK = 6.7) respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Fe Uptake Mechanism in Fe-Efficient Cucumber Roots

Fe-efficient plants respond to iron stress both by morphological and physiological modifications. In roots of a Fe-efficient plant (Cucumis sativus L.) grown in the presence or in the absence of iron, the capacity to acidify the external medium, change in the transmembrane electrical potential, and the ATPase activity have been determined. Roots from plants grown in the absence of iron showed a great capacity to acidify the external medium, a higher transmembrane electrical potential difference (-145 millivolts, versus -105 millivolts), and a higher ATPase activity (+30%). The administration of Fe(2+), but not Fe(3+), caused a block of the acidification capacity, a great decrease in the transmembrane electrical potential difference in root cells, and a large inhibition of the ATPase activity of isolated microsomal membrane vesicles.

Power transmission along biological membranes

Hypothesis on long-distance power transmission along extended energy-transducing membranes (Skulachev, 1969, 1971, 1980), has been experimentally proven in four different systems, namely, (i) trichomes of filamentous cyanobacterium Phormidium uncinatum; (ii) filamentous mitochondria and mitochondrial network in fibroblasts; (iii) clusters of roundish heart muscle mitochondria interconnected with mitochondrial junctions; (iv) mixed animal cell cultures interconnected with gap junctions. In all cases, energy was shown to be transmitted in the form of a transmembrane electric potential difference. The transmission occurred for distances as long as several tens of micrometers. Since the (a) delta-muH-bearing cytoplasmic membrane of cyanobacteria and the inner mitochondrial membrane and (b) delta-muNa-bearing outer animal cell membrane were found to be competent in such an effect, one may assume that the power transmission is a fundamental function of extended membrane systems. This mechanism can be used at the intracellular level (mitochondrial) as well as at the supracellular level (cytoplasmic and outer cell membranes). Studies on the possible involvement of membranes in lateral transport of oxygen, ions, fatty acids and membrane proteins seem to hold good promise.

Electrogenesis associated with proton transfer in the reaction center protein of the purple bacterium Rhodobacter sphaeroides

Electrogenic events in the photosynthetic reaction center complex (RC), accompanying single- and two-electron reduction of the secondar quinone acceptor Q b, were investigated. In the presence of inhibitors of electron transfer via the bc 1-complex, the kinetics of formation of the transmembrane electric potential difference induced by two successive light flashes exhibit a few phases. Besides the fast phase A which is due to the charge separation between the bacteriochlorophyll dimer P and primary quinone acceptor q a, two slower atrazine-sensitive phases, BI and BII, were observed. Phase BI is suggested to be due to proton transfer between the amino acid residues of the reaction center protein, and phase BII due to proton uptake during the second flash-induced formation of ubiquinol. A possible model of electrogenesis in the acceptor moiety of the RC is discussed.

Alkaline secretion by Necturus proximal duodenal mucosa

Proximal duodenum from the amphibian Necturus was stripped of muscle layers and the mucosa was mounted as a tube for studies of alkali transport or as a flat sheet for intracellular impalement by voltage-sensitive glass micro-electrodes. The mucosa alkalinized the unbuffered luminal perfusate at a high rate (3.4 muequiv. cm-1 h-1) and developed a transepithelial electric potential difference of 5.7 mV (lumen negative). Transport was inhibited by 2,4-dinitrophenol (10(-4) M) and by furosemide (10(-3) M) and SITS (10(-3) M) on the seros but not on the mucosal side, indicating dependence on tissue metabolism and on serosal membrane Cl-/HCO3- exchange. Prostaglandin E2 (10(-7)-10(-5) M) and dibutyryl cyclic AMP (10(-6)-10(-4) M) had no effects on the secretion or transepithelial electrical potential difference. removal of serosal HCO3- decreased luminal alkalinization by 75%, indicating a contribution by passive migration of HCO3- and/or a dependence of transcellular transport on the nutrient supply of this ion. Administration of HCO3- (17.8 mM) to the luminal perfusate affected neither the transepithelial nor transmembrane electrical potential differences nor the resistance ratio. It is thus unlikely that the luminal membrane possesses any major HCO3- conductance.

Kinetic analysis of electrogenic 2 Na+-1 H+ antiport in crustacean hepatopancreas

Na+ uptake by short-circuited brush-border membrane vesicles of the hepatopancreatic epithelium from the freshwater prawn Macrobrachium rosenbergii was Cl- independent, amiloride sensitive, and stimulated by a transmembrane proton gradient ([H+]i greater than [H+]o). Na+ influx (3-s uptake) was a sigmoidal function of [Na]o (2.5-150 mM), when pHi = 6.0, pHo = 8.0, and followed the Hill equation for binding cooperativity [maximal Na+ influx (Jm) = 140.6 nmol mg-1s-1; affinity constant (K') = 82.2 mM Na+; Hill coefficient (n) = 2.07]. Influx kinetic analyses at physiological conditions suggested two external cation-binding sites shared by Na+ and H+ (proton dissociation constant Pk1 = 5.7; Pk2 = 4.0) and a single internal cation site used only by H+ (Pk = 6.5). Amiloride was a competitive inhibitor of Na+ transport at both external binding sites (Ki1 = 50 microM; Ki2 = 1,520 microM). Electrogenic Na+-H+ exchange by these vesicles was demonstrated using an equilibrium-shift method of analysis and a transmembrane electrical potential difference as the only driving force for transport. In addition, electrogenic net Na+ influx (3-s uptake) was observed in vesicles loaded with 5 mM 22Na at pH 7.0 and exposed to media containing several 22Na or proton concentrations. Results suggest the following exchange model: low [Na]o, (1 Na+ and 1 H+)-1 H+; high [Na+]o, 2 Na+-1 H+. This antiport mechanism may account for two major functional operations of the gastrointestinal tract in these animals: 1) proton secretion against considerable concentration gradients leading to stomach luminal acidification, and 2) Na+ absorption from lumen to cytoplasm potentially making a significant contribution to organismic ion balance.

Na-dependent L-glutamate transport by eel intestinal BBMV: role of K+ and Cl-

L-[3H]glutamate uptake into eel (Anguilla anguilla) intestinal brush-border membrane vesicles (BBMV) was a sigmoidal function of extravesicular Na, suggesting that two or more cations accompanied the amino acid during transport. L-[3H]glutamate influx illustrated the following kinetic constants: apparent membrane binding affinity (Kapp) = 0.80 +/- 0.12 mM; influx velocity (Jmax) = 2.61 +/- 0.31 nmol.mg protein-1.min-1; and permeability coefficient (P) = 0.65 +/- 0.10 microliters.mg protein-1. min-1. Results from the imposition of diffusion potentials across vesicle membranes using K-valinomycin or H-carbonyl-cyanide p-chloromethoxyphenylhydrazone suggested that Na-dependent L-glutamate transport was sensitive to transmembrane electrical potential difference. Extravesicular aspartate was a competitive inhibitor of L-[3H]glutamate influx [inhibitory constant (Ki) = 0.28 +/- 0.04 mM]. Intravesicular K and extravesicular Cl ions enhanced maximal amino acid influx and transient L-glutamate accumulation against a concentration gradient (overshoot). Intravesicular K reduced the Kapp of the membrane to L-glutamate, whereas extravesicular Cl increased L-glutamate Jmax. A model for L-[3H]glutamate transport is suggested involving the cotransport of at least two Na and one L-glutamate that is activated by one intravesicular K ion and at least two extravesicular Cl ions.

Protonmotive force in Brevibacterium flavum: the lack of intracellular pH homeostasis

Brevibacterium flavum 22LD-P cells were shown to maintain a transmembrane pH gradient (ΔpH) from 0.6 to 1.8–2 units and a transmembrane electric potential difference (ΔΨ) from 0 to 200 mV depending on the pH and ionic composition of the incubation medium, grwoth substrate and concentration of cells. ΔΨ decreased from 120–140 mV to 0 when medium pH was lowered from neutral to 5.0–5.5 and increased to 180–200 mV when medium pH was raised to 8–9 in cells utilizing acetate or endogenous substrate. Cells growing on sucrose, kept ΔΨ around 100–120 mV at neutral as well as acidic medium pH. Intracellular pH in the acetate utilizing or endogenously respiring cells was maintained with the range of 8.9 to 5.5 at medium pH ranging from 9.1 to 4.0, respectively. Sucrose grown cells were able to maintain a more stable intracellular pH. Endogenously respiring cells in potassium phosphate buffer at high biomass concentrations maintained larger ΔpH and relatively smaller ΔΨ, than the same cells in diluted suspensions. Cells in sodium phosphate buffer possessed larger ΔΨ and almost no ΔpH, but ΔΨ was still dependent on biomass concentration.

The electrogenic proton/hexose carrier in the plasmalemma of Chenopodium rubrum suspension cells: effects of Δc, ΔpH and Δψ on hexose exchange diffusion

The hexose carrier in the plasmalemma of a higher plant suspension cell ( Chenopodium rubrum L.) catalyzes not only electrogenic proton/hexose symport, but also electroneutral exchange diffusion of hexoses. The transport process is studied mainly through short-term efflux disturbance of labelled 3-O- methyl- d-glucose (3-OMG). This novel method singles out the transport system in situ and reliably detects small (>5%) changes of unidirectional hexose fluxes. The three driving forces for the electrogenic net uptake of hexose are the gradients of substrate (hexose), cosubstrate (H +) and the electric potential difference across the plasmalemma. Increasing substrate concentrations (outside or inside) led to increased unidirectional fluxes into the compartment where the substrate concentration was increased (e.g., trans stimulation of 3-OMG efflux by external hexose); however, an increase of the external H + concentration at non-saturating external hexose concentrations led to a trans inhibition of 3-OMG efflux. Depolarization of the transmembrane electric potential difference without influencing the surface potential led to only a small increase in hexose efflux at low hexose concentrations and had no measurable effect at saturating hexose concentrations. However, depolarization mainly due to a decrease of the surface and Donnan potentials mimicked the effect of alkalinization. These findings agree with a model based on a negatively charged carrier showing random-order binding of substrate and cosubstrate on the outside of the plasmalemma. It is not necessary to assume a recycling translocation step of the empty substrate-binding site. The low-voltage sensitivity of the hexose efflux observed is discussed with regard to possible electric potential profiles at the site of transport.

Kinetics of d-glucose transport across the intestinal brush-border membrane of the cat

1. 1. d-glucose transport across the intestinal brush-border membrane of the cat, a carnivorous animal, was investigated using isolated brush-border membrane vesicles (BBMV). Kinetic experiments were performed under zero-trans conditions (initial [Na +] in and [Gluc] in = 0) with the transmembrane electrical potential difference clamped to zero. 2. 2. d-glucose uptake by the BBMV was strongly stimulated by an inwardly directed Na +-gradient. Uptake under Na +-free conditions seemed to occur by simple diffusion. 3. 3. The apparent kinetic constants ( V max, K m) of Na +-dependent d-glucose transport were computed by forcing initial uptake rates at 0.002–10.0 mmol/1 d-glucose to either a Michaelis-Menten type equation with a single or with two carrier-mediated components. 4. 4. Best fit of the experimental data was obtained with the two-component model indicating the existence of two Na +-dependent carrier-mediated mechanisms. System 1 and system 2 differ with respect to the transport velocity as well as the substrate affinity constants with V max being 2.5-fold and K m being 5-fold higher for system 1 compared with system 2.