Inside-negative Membrane Potential Research Articles

Lipophilic ion aromaticity is not important for permeability across lipid membranes

To clarify the contribution of charge delocalization in a lipophilic ion to the efficacy of its permeation through a lipid membrane, we compared the behavior of alkyl derivatives of triphenylphosphonium, tricyclohexylphosphonium and trihexylphosphonium both in natural and artificial membranes. Exploring accumulation of the lipophilic cations in response to inside-negative membrane potential generation in mitochondria by using an ion-selective electrode revealed similar mitochondrial uptake of butyltricyclohexylphosphonium (C4TCHP) and butyltriphenylphosphonium (C4TPP). Fluorescence correlation spectroscopy also demonstrated similar membrane potential-dependent accumulation of fluorescein derivatives of tricyclohexyldecylphosphonium and decyltriphenylphosphonium in mitochondria. The rate constant of lipophilic cation translocation across the bilayer lipid membrane (BLM), measured by the current relaxation method, moderately increased in the following sequence: trihexyltetradecylphosphonium ([P6,6,6,14]) < triphenyltetradecylphosphonium (C14TPP) < tricyclohexyldodecylphosphonium (C12TCHP). In line with these results, measurements of the BLM stationary conductance indicated that membrane permeability for C4TCHP is 2.5 times higher than that for C4TPP. Values of the difference in the free energy of ion solvation in water and octane calculated using the density functional theory and the polarizable continuum solvent model were similar for methyltriphenylphosphonium, tricyclohexylmethylphosphonium and trihexylmethylphosphonium. Our results prove that both cyclic and aromatic moieties are not necessary for lipophilic ions to effectively permeate through lipid membranes.

Comparison of Methods for Estimating Unbound Intracellular-to-Medium Concentration Ratios in Rat and Human Hepatocytes Using Statins

It is essential to estimate concentrations of unbound drugs inside the hepatocytes to predict hepatic clearance, efficacy, and toxicity of the drugs. The present study was undertaken to compare predictability of the unbound hepatocyte-to-medium concentration ratios (Kp,uu) by two methods based on the steady-state cell-to-medium total concentration ratios at 37°C and on ice (Kp,uu,ss) and based on their initial uptake rates (Kp,uu,V0). Poorly metabolized statins were used as test drugs because of their concentrative uptake via organic anion-transporting polypeptides. Kp,uu,ss values of these statins provided less interexperimental variation than the Kp,uu,V0 values, because only data at longer time are required for Kp,uu,ss Kp,uu,V0 values for pitavastatin, rosuvastatin, and pravastatin were 1.2- to 5.1-fold Kp,uu,ss in rat hepatocytes; Kp,uu,V0 values in human hepatocytes also tended to be larger than corresponding Kp,uu,ss To explain these discrepancies, theoretical values of Kp,uu,ss and Kp,uu,V0 were compared with true Kp,uu (Kp,uu,true), considering the inside-negative membrane potential and ionization of the drugs in hepatocytes and medium. Membrane potentials were approximately -30 mV in human hepatocytes at 37°C and almost abolished on ice. Theoretical equations considering the membrane potentials indicate that Kp,uu,ss values for the statins are 0.85- to 1.2-fold Kp,uu,true, whereas Kp,uu,V0 values are 2.2- to 3.1-fold Kp,uu,true, depending on the ratio of the passive permeability of the ionized to nonionized forms. In conclusion, Kp,uu,ss values of anions are similar to Kp,uu,true when the inside-negative membrane potential is considered. This suggests that Kp,uu,ss is preferable for estimating the concentration of unbound drugs inside the hepatocytes.

Probing the Cl−-pumping photocycle of pharaonis halorhodopsin: Examinations with bacterioruberin, an intrinsic dye, and membrane potential-induced modulation of the photocycle

Halorhodopsin (HR) functions as a light-driven inward Cl− pump. The Cl− transfer process of HR from Natronomonas pharaonis (NpHR) was examined utilizing a mutant strain, KM-1, which expresses large amount of NpHR in a complex with the carotenoid bacterioruberin (Brub). When Cl− was added to unphotolyzed Cl−-free NpHR–Brub complex, Brub caused the absorption spectral change in response to the Cl− binding to NpHR through the altered electrostatic environment and/or distortion of its own configuration. During the Cl−-puming photocycle, on the other hand, oppositely directed spectral change of Brub appeared during the O intermediate formation and remained until the decay of the last intermediate NpHR′. These results indicate that Cl− is released into the cytoplasmic medium during the N to O transition, and that the subsequent NpHR′ still maintains an altered protein conformation while another Cl− already binds in the vicinity of the Schiff base. Using the cell envelope vesicles, the effect of the interior negative membrane potential on the photocycle was examined. The prominent effect appeared in the shift of the N–O quasi-equilibrium toward N, supporting Cl− release during the N to O transition. The membrane potential had a much larger effect on the Cl− transfer in the cytoplasmic half channel compared to that in the extracellular half channel. This result may reflect the differences in dielectric constants and/or lengths of the pathways for Cl− transfers during N to O and O to NpHR′ transitions.

Toward high-content screening of mitochondrial morphology and membrane potential in living cells

Mitochondria are double membrane organelles involved in various key cellular processes. Governed by dedicated protein machinery, mitochondria move and continuously fuse and divide. These “mitochondrial dynamics” are bi-directionally linked to mitochondrial and cell functional state in space and time. Due to the action of the electron transport chain (ETC), the mitochondrial inner membrane displays a inside-negative membrane potential (Δψ). The latter is considered a functional readout of mitochondrial “health” and required to sustain normal mitochondrial ATP production and mitochondrial fusion. During the last decade, live-cell microscopy strategies were developed for simultaneous quantification of Δψ and mitochondrial morphology. This revealed that ETC dysfunction, changes in Δψ and aberrations in mitochondrial structure often occur in parallel, suggesting they are linked potential targets for therapeutic intervention. Here we discuss how combining high-content and high-throughput strategies can be used for analysis of genetic and/or drug-induced effects at the level of individual organelles, cells and cell populations.This article is part of a Directed Issue entitled: Energy Metabolism Disorders and Therapies.

Role of Thr218 in the Light-Driven Anion Pump Halorhodopsin from Natronomonas pharaonis

Halorhodopsin (HR) is an inward-directed light-driven halogen ion pump, and NpHR is a HR from Natronomonas pharaonis. Unphotolyzed NpHR binds halogen ion in the vicinity of the Schiff base, which links retinal to Lys256. This halogen ion is transported during the photocycle. We made various mutants of Thr218, which is located one half-turn up from the Schiff base to the cytoplasm (CP) channel, and analyzed the photocycle using a sequential irreversible model. Four photochemically defined intermediates (P(i), i = 1-4) were adequate to describe the photocycle. The third component, P₃, was a quasi-equilibrium complex between the N and O intermediates, where a N ↔ O + Cl⁻ equilibrium was attained. The K(d,N↔O) values of this equilibrium for various mutants were determined, and the value of Thr (wild type) was the highest. The partial molar volume differences between N and O, ΔV(N→O), were estimated from the pressure dependence of K(d,N↔O). A comparison between K(d,N↔O) and ΔV(N→O) led to the conclusion that water entry by the F-helix opening at O may occur, which may increase K(d,N↔O). For some mutants, however, large ΔV(N→O) values were found, whereas the K(d,N↔O) values were small. This suggests that the special coordination of a water molecule with the OH group of Thr is necessary for the increase in K(d,N↔O). Mutants with a small K(d,N↔O) showed low pumping activities in the presence of inside negative membrane potential, while the mutant activities were not different in the absence of membrane potential. The effect of the mutation on the pumping activities is discussed.

3H‐D‐glucose transport is both Na+‐ and K+‐dependent in the marine shrimp, Litopenaeus setiferus

This study reports the occurrence of a brush border 3H‐D‐glucose transporter in the digestive tract of a marine shrimp that is similarly stimulated by both Na+ and K+. Hepatopancreatic brush border membrane vesicles loaded with 300 mM mannitol exhibited 0.1 mM 3H‐glucose uptake overshoots, that were membrane‐potential‐ sensitive, when incubated in media containing either 150 mM NaCl or KCl. Sugar influxes (15 sec uptakes) in both NaCl or KCl were significantly (p &lt; 0.05) inhibited by 5 mM phloridzin. 3H‐glucose (0.1 mM) influxes in media containing concentrations of NaCl or KCl from 0 to 400 mM were hyperbolic functions of the salt concentrations with both Km‐ and Jmax –values generally higher for KCl than for NaCl. Significant increases (p &lt; 0.05) in Km‐ and Jmax‐values occurred in both media in the presence of a membrane potential difference (inside negative). 3H‐glucose influx was a hyperbolic function of [glucose] from 0.1 to 5.0 mM in the presence of either 150 mM NaCl or KCl. Influx kinetic constants (Km and Jmax) were higher in NaCl than in KCl, and imposed inside‐negative membrane potentials decreased the kinetic constants in both instances. Results suggest that sugar absorption in shrimp may be largely K+‐dependent during juvenile herbivory and Na+‐dependent in adult omnivory/carnivory. Supported by USDA agriculture and food initiative competitive grant no. 2010‐65206‐20617.

Electrophysiological Characterization of the Polyspecific Organic Cation Transporter Plasma Membrane Monoamine Transporter

Plasma membrane monoamine transporter (PMAT) is a polyspecific organic cation (OC) transporter that transports a variety of endogenous biogenic amines and xenobiotic cations. Previous radiotracer uptake studies showed that PMAT-mediated OC transport is sensitive to changes in membrane potential and extracellular pH, but the precise role of membrane potential and protons on PMAT-mediated OC transport is unknown. Here, we characterized the electrophysiological properties of PMAT in Xenopus laevis oocytes using a two-microelectrode voltage-clamp approach. PMAT-mediated histamine uptake is associated with inward currents under voltage-clamp conditions, and the currents increased in magnitude as the holding membrane potential became more negative. A similar effect was also observed for another cation, nicotine. Substrate-induced currents were largely independent of Na+ but showed strong dependence on membrane potential and pH of the perfusate. Detailed kinetic analysis of histamine uptake revealed that the energizing effect of membrane potentials on PMAT transport is mainly due to an augmentation of Imax with little effect on K0.5. At most holding membrane potentials, Imax at pH 6.0 is approximately 3- to 4-fold higher than that at pH 7.5, whereas K0.5 is not dependent on pH. Together, these data unequivocally demonstrate PMAT as an electrogenic transporter and establish the physiological inside-negative membrane potential as a driving force for PMAT-mediated OC transport. The important role of membrane potential and pH in modulating the transport activity of PMAT toward OCs suggests that the in vivo activity of PMAT could be regulated by pathophysiological processes that alter physiological pH or membrane potential.

Characterization of Human OCT1-Mediated Transport of DAPI as a Fluorescent Probe Substrate

The present study was conducted to assess the functional characteristics of human organic cation transporter 1 (hOCT1) for the transport of 4′,6-diamidino-2-phenylindol (DAPI), a fluorescent compound that may be used as a probe substrate for rapid assays of its functionality. The specific uptake of DAPI by hOCT1 heterologously introduced into Madin–Darby canine kidney II cells by stable transfection was found to be, when assessed by DAPI-derived fluorescence intensity, rapid and saturable with a Michaelis constant of 8.94 µM, indicating that DAPI is a good substrate of hOCT1. The specific uptake of DAPI was insensitive to the membrane potential and extracellular pH, indicating a mode of operation different from that for typical cationic substrates such as tetraethylammonium (TEA), for which hOCT1 has been suggested to be driven by an inside-negative membrane potential and favor higher pH for optimal operation. However, many organic cations were found to inhibit the specific DAPI uptake with extents well correlated with those of inhibition of the specific uptake of [14C]TEA, indicating comparable performances of both substrates as probes in identifying inhibitors. Thus, DAPI can be an alternative probe substrate that enables fluorometric rapid assays of the functionality of hOCT1. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:4006–4012, 2011

Substrate-dependent proton antiport in neurotransmitter:sodium symporters

SUMMARYNeurotransmitter:sodium symporters (NSS), targets for psychostimulants and therapeutic drugs, play a critical role in neurotransmission. Whereas eukaryotic NSS exhibit Cl−-dependent transport, bacterial NSS feature Cl−-independent substrate transport. Recently we showed in LeuT and Tyt1 that mutation of an acidic side chain near one of the Na+-binding sites renders substrate binding and/or transport Cl− dependent. We reasoned that the negative charge - provided either by Cl− or by the transporter itself - is required for substrate translocation. Here we show that Tyt1 reconstituted in proteoliposomes is strictly dependent on the Na+ gradient and is stimulated by an inside negative membrane potential and by an inversely-oriented H+ gradient. Remarkably, Na+/substrate symport elicited H+ efflux, indicative of Na+/substrate symport-coupled H+ antiport. Mutations that render the transport phenotype Cl−-dependent essentially abolish the pH dependence. We propose unifying features of charge balance by all NSS members with similar mechanistic features but with different molecular solutions.

Stimuli-responsive polyguanidino-oxanorbornene membrane transporters as multicomponent sensors in complex matrices.

We introduce guanidinium-containing synthetic polymers based on polyguanidino-oxanorbornenes (PGONs) as anion transporters in lipid bilayers that can be activated and inactivated by chemical stimulation. According to fluorogenic anion export experiments with vesicles, PGON transporters are most active in neutral bilayers near their phase transition, with EC50’s in the nanomolar range. Six times higher effective transporter concentrations were measured with aminonaphthalene-1,3,6-trisulfonate than with 5(6)-carboxyfluorescein, demonstrating the importance of anion binding for transport and excluding nonspecific efflux. Negative surface potentials efficiently annihilate transport activity, while inside-negative membrane potentials slightly increase it. These trends demonstrate the functional importance of counterions to hinder the binding of hydrophilic counterions and to minimize the global positive charge of the transporter−counterion complexes. Strong, nonlinear increases in activity with polymer length reveal a significant polymer effect. Overall, the characteristics of PGONs do not match those of similar systems (for example, polyarginine) and hint toward an interesting mode of action, clearly different from nonspecific leakage caused by detergents. The activity of PGONs increases in the presence of amphiphilic anions such as pyrenebutyrate (EC50 = 70 μM), while several other amphiphilic anions tested were inactive. PGONs are efficiently inactivated by numerous hydrophilic anions including ATP (IC50 = 150 μM), ADP (IC50 = 460 μM), heparin (IC50 = 1.0 μM), phytate (IC50 = 0.4 μM), and CB hydrazide (IC50 = 26 μM). The compatibility of this broad responsiveness with multicomponent sensing in complex matrices is discussed and illustrated with lactate sensing in sour milk. The PGON lactate sensor operates together with lactate oxidase as a specific signal generator and CB hydrazide as an amplifier for covalent capture of the pyruvate product as CB hydrazone (IC50 = 1.5 μM).

Function and Subunit Interactions of the N-terminal Domain of Subunit a (Vph1p) of the Yeast V-ATPase

The vacuolar (H+)-ATPases (V-ATPases) are ATP-dependent proton pumps that operate by a rotary mechanism in which ATP hydrolysis drives rotation of a ring of proteolipid subunits relative to subunit a within the integral V(0) domain. In vivo dissociation of the V-ATPase (an important regulatory mechanism) generates a V(0) domain that does not passively conduct protons. EM analysis indicates that the N-terminal domain of subunit a approaches the rotary subunits in free V(0), suggesting a possible mechanism of silencing passive proton transport. To test the hypothesis that the N-terminal domain inhibits passive proton flux by preventing rotation of the proteolipid ring in free V(0), factor Xa cleavage sites were introduced between the N- and C-terminal domains of subunit a (the Vph1p isoform in yeast) to allow its removal in vitro after isolation of vacuolar membranes. The mutant Vph1p gave rise to a partially uncoupled V-ATPase complex. Cleavage with factor Xa led to further loss of coupling of proton transport and ATP hydrolysis. Removal of the N-terminal domain by cleavage with factor Xa and treatment with KNO3 and MgATP did not, however, lead to an increase in passive proton conductance by free V(0), suggesting that removal of the N-terminal domain is not sufficient to facilitate passive proton conductance through V(0). Photoactivated cross-linking using the cysteine reagent maleimido benzophenone and single cysteine mutants of subunit a demonstrated the proximity of specific sites within the N-terminal domain and subunits E and G of the peripheral stalk. These results suggest that a localized region of the N-terminal domain (residues 347-369) is important in anchoring the peripheral stator in V1V0.

Transport of drugs in the kidney by the human organic cation transporter, OCT2 and its genetic variants

The human organic cation transporter 2 (OCT2, SLC22A2) is a multispecific transporter of organic cations, including many clinically used drugs. OCT2 is primarily responsible for the uptake of organic cations across the basolateral membrane of renal tubular epithelial cells and is considered a major transporter in the active secretion of organic cations in the kidney. Uptake of organic cations by OCT2 is driven by the inside-negative membrane potential and is pH-sensitive. Regulation of OCT2 at the transcriptional level by steroid hormones and at the protein level by various protein kinases has been described. Several human genetic variants in the coding region of OCT2 have been identified and functionally characterized, including both polymorphic and rare variants. A variety of structurally diverse compounds have been shown to interact with OCT2, including endogenous compounds, drugs, and dietary supplements.

Purification and Functional Reconstitution of the Rat Organic Cation Transporter OCT1

The rat organic cation transporter rOCT1 with six histidine residues added to the C-terminus was expressed in Sf9 insect cells, and expression of organic cation transport was demonstrated. To purify rOCT1 protein, Sf9 cells were lysed with 1% (w/v) CHAPS [3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate], centrifuged, and subjected to sequential affinity chromatography using lentil-lectin Sepharose and nickel(II)-charged nitrilotriacetic acid-agarose. This procedure yielded approximately 70 microg of purified rOCT1 protein from 10 standard culture plates. Using a freeze-thaw procedure, purified rOCT1 was reconstituted into proteoliposomes formed from phosphatidylcholine, phosphatidylserine, and cholesterol. Proteoliposomes exhibited uptake of [3H]-1-Methyl-4-phenylpyridinium ([3H]MPP) that was inhibited by quinine and stimulated by an inside-negative membrane potential. MPP uptake was saturable with an apparent K(m) of 30 +/- 17 microM. MPP uptake (0.1 microM) was inhibited by tetraethylammonium, tetrabutylammonium, and tetrapentylammonium with IC50 values of 197 +/- 11, 19 +/- 1, and 1.8 +/- 0.03 microM, respectively. With membrane potential clamped to 0 mV using valinomycin in the presence of 100 mM potassium on both sides of the membrane, uptake of 0.1 microM MPP was trans stimulated 3-fold by 2.5 mM intracellular choline, and efflux of 0.1 microM MPP was trans stimulated 4-fold by 9.5 mM extracellular choline. The data show that rOCT1 is capable and sufficient to mediate transport of organic cations. The observed trans stimulation under voltage-clamp conditions shows that rOCT1 operates as a transporter rather than a channel. Purification and reconstitution of functional active rOCT1 protein is an important step toward the biophysical characterization and crystallization.

Differential expression of Na+/D-glucose cotransport in isolated cells of Marsupenaeus japonicus hepatopancreas.

D-Glucose absorptive processes at the gastrointestinal tract of decapod crustaceans are largely under-investigated. We have studied Na(+)-dependent D-glucose transport (Na(+)/D-glucose cotransport) in the hepatopancreas of the Kuruma prawn, Marsupenaeus japonicus, using both brush-border membrane vesicles and purified R and B hepatopancreatic cell suspensions. As assessed by brush-border membrane vesicle studies, Na(+)/D-glucose cotransport was inhibited by phloridzin and responsive to the (inside negative) membrane potential. Furthermore, it was strongly activated by protons (although only in the presence of an inside-negative membrane potential), which correlates with the fact that the lumen of crustacean hepatopancreatic tubules is acidic. When assayed in purified R and B cell suspensions, Na(+)/D-glucose cotransport activity was restricted to B cells only. Mab 13, a monoclonal antibody recognizing an 80- to 85-KDa protein at the brush-border membrane location, inhibited Na(+)/D-glucose cotransport in brush-border membrane vesicles as well as in enriched B cell suspensions. Primers designed after comparison of highly homologous regions of various mammalian sodium-glucose transporter) nucleotide sequences failed to produce RT-PCR amplification products from Kuruma prawn hepatopancreatic RNA. The molecular nature of this Na(+)/D-glucose cotransport system is still to be established.

PH dependence of Na+/myo-inositol cotransporters in rat thick limb cells

To balance medullary interstitium hypertonicity generated by transepithelial NaCl absorption, medullary thick ascending limb (MTAL) cells accumulate myo-inositol (MI). Expression of Na+-MI cotransporter (SMIT) mRNA in TAL is correlated with the NaCl absorption rate. Our present study aimed to determine the plasma membrane location and functional properties of the Na+-MI cotransporter in MTAL cells. Preparation of basolateral (BLMV) and luminal (LMV) membrane vesicles were simultaneously isolated from purified rat MTAL suspension, and uptake of [3H]myo-inositol ([3H]MI) was used to assess Na+-MI cotransport activity. In the presence of an inside-negative membrane potential, imposing an inwardly-directed Na+-gradient versus tetramethylammonium (TMA) stimulated the initial [3H]MI uptake in BLMV and LMV. Phlorizin inhibited Na+ gradient-dependent initial [3H]MI uptake in both preparations, with IC50 values of 565 and 29 micromol/L in BLMV and LMV, respectively. 2-0,C-methylene myo-inositol (MMI), a competitive inhibitor of MI transport, only inhibited the BLMV Na+-MI cotransporter. Phlorizin-sensitive Na+ gradient-dependent initial [3H]MI uptake showed Michaelis-Menten kinetics in both preparations, with similar Vmax but different Km values of 51 and 107 micromol/L in BLMV and LMV, respectively. Finally, BLMV but not LMV Na+-MI cotransporter exhibited a marked pH dependence with sigmoidal patterns of activation, as intravesicular pH (pHi) was decreased from 8.0 to 6.0 at extravesicular pH (pHe) 8.0, and as pHe was increased from 6.0 to 8.0 at pHi 6.0. Maximal activation was observed at pHi 6.5 and pHe 7.5. In rat MTAL cells, Na+-MI cotransporter activity is present in both BLM and LM, and has markedly different functional properties, indicating the presence of distinct transporters. Basolateral Na+-MI cotransporter activity is maximal at physiological pH values of MTAL cells and interstitium, and a powerful modulation of the transporter activity may be exerted by pHe and pHi.

Electrolyte Composition of Mink (Mustela vison) Erythrocytes and Active Cation Transporters of the Cell Membrane

Red blood cells from mink (Mustela vison) were characterized with respect to their electrolyte content and their cell membranes with respect to enzymatic activity for cation transport. The intra- and extracellular concentrations of Na+, K+, Cl-, Ca2+ and Mg2+ were determined in erythrocytes and plasma, respectively. Plasma and red cell water content was determined, and molal electrolyte concentrations were calculated. Red cells from male adult mink appeared to be of the low-K+, high-Na+ type as seen in other carnivorous species. The intracellular K+ concentration is slightly higher than the extracellular one and the plasma-to-cell chemical gradient for Na+ is weak, though even the molal concentrations may differ significantly. Consistent with the high intracellular Na+ and low K+ concentrations, a very low or no ouabain-sensitive Na+,K+-ATPase activity and no K+-activated pNPPase activity were found in the plasma membrane fraction from red cells. The Cl- and Mg2+ concentrations expressed per liter cell water were significantly higher in red cells than in plasma whereas the opposite was the case with Ca2+. The distribution of Cl- thus does not seem compatible with an inside-negative membrane potential in mink erythrocytes. In spite of a steep calcium gradient across the red cell membrane, neither a calmodulin-activated Ca2+-ATPase activity nor an ATP-activated Ca2+-pNPPase activity were detectable in the plasma membrane fraction. The origin of a supposed primary Ca2+ gradient for sustaining of osmotic balance thus seems uncertain.

Characterisation of the H(+)/peptide cotransporter of eel intestinal brush-border membranes.

H(+)/peptide cotransport in brush-border membrane vesicles (BBMVs) from eel (Anguilla anguilla) intestine was studied by measuring d-[(3)H]-phenylalanyl-l-alanine uptake and by monitoring peptide-dependent intravesicular acidification using the pH-sensitive dye Acridine Orange. d-[(3)H]-phenylalanyl-l-alanine influx was greatly stimulated by an inside-negative membrane potential and enhanced by an inwardly directed H(+) gradient. In parallel, vesicular H(+) influx was significantly increased in the presence of extravesicular d-phenylalanyl-l-alanine or a series of glycyl and l-prolyl peptides. H(+)/peptide cotransport displayed saturable kinetics involving a single carrier system with apparent substrate affinities of 0.9-2.6 mmol l(-1) depending on the particular peptide. All substrates tested competed with this system. Pre-incubation of BBMVs with dipeptides prevented diethylpyrocarbonate inhibition of transport activity, suggesting that the substrates mask histidine residues involved in the catalytic function of the transporter. Using human PepT1-specific primers, a reverse transcription-polymerase chain reaction (RT-PCR) signal was detected in eel intestine. Our results suggest that, in eel intestine, a brush-border membrane 'low-affinity'-type H(+)/peptide cotransport system is present that shares kinetic features with the mammalian intestinal PepT1-type transporters.

Sulfate/bicarbonate antiport by lobster hepatopancreatic basolateral membrane vesicles

Sulfate transport across plasma membranes has been described in a wide variety of organisms and cell types including gastrointestinal epithelia. Sulfate transport can be coupled to proton or sodium symport or antiport processes involving a variety of anions. It had been previously observed in lobster hepatopancreas that sulfate could be secreted, however, the mechanisms for this potential secretory process had not been fully described. Therefore, the present study was done to delineate one of the potential processes for this transport. Purified basolateral membrane vesicles (BLMV) were prepared from lobster hepatopancreas by osmotic disruption and discontinuous sucrose gradient centrifugation. Transport of (35S) sulfate into BLMV was stimulated by an outwardly directed bicarbonate gradient compared with gluconate-loaded vesicles. An inside-positive membrane potential (valinomycin and K+) stimulated sulfate/bicarbonate exchange; whereas an inside-negative membrane potential was inhibitory. Sulfate/sulfate exchange was not affected by alterations of transmembrane potential difference. External protons stimulated sulfate/bicarbonate exchange although proton gradients had no effect on sulfate/bicarbonate exchange in the BLMV. The stilbenes, 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS) and 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), strongly inhibited sulfate/bicarbonate exchange. These results suggest that sulfate/bicarbonate exchange in hepatopancreatic BLMV occurred by an electrogenic carrier mechanism exhibiting a 1:1 flux ratio. The possible physiological role of this antiport process in sulfate transport across the crustacean hepatopancreas is discussed. J. Exp. Zool. 284:158–167, 1999. © 1999 Wiley-Liss, Inc.

Spermine and Arcaine Block and Permeate N-Methyl- d-Aspartate Receptor Channels

Polyamines such as spermine are thought to be endogenous regulators of NMDA ( N-methyl- d-aspartate)-type glutamate receptors. Polyamine block of NMDA receptors was studied in excised outside-out patches from rat hippocampal neurons and Xenopus oocytes expressing recombinant receptors. Extracellular spermine and arcaine reduced NMDA single-channel conductance in a voltage-dependent manner, with partial relief of block evident at large inside negative membrane potentials. Reducing extracellular Na + concentration increased the apparent affinities for spermine and arcaine, indicating strong interaction between spermine and permeant ions. Internal spermine also blocked NMDA channels in a voltage-dependent manner, with relief of block evident at large inside positive potentials. The Woodhull model of channel block by an impermeant ion adequately described the actions of external spermine from −60 to +60 mV, but failed for more negative potentials. Eyring rate theory for a permeable blocker with two barriers and one binding site adequately described the voltage-dependent block and relief from block by both external and internal spermine over the range of −120 to +60 mV. These findings indicate that polyamines block and permeate neuronal NMDA receptor channels from the extracellular and intracellular sides, although sensitivity to internal spermine is probably too low to be physiologically relevant.

Accurate flow cytometric membrane potential measurement in bacteria using diethyloxacarbocyanine and a ratiometric technique.

Membrane potential (MP) plays a critical role in bacterial physiology. Existing methods for MP estimation by flow cytometry are neither accurate nor precise, due in part to the heterogeneity of size of the particles analyzed. The ratio of a size- and MP-sensitive measurement, and an MP-independent, size-sensitive measurement, should provide a better estimate of MP. Flow cytometry and spectrofluorometry were used to detect red (488 --> 600 nm) fluorescence associated with aggregates of diethyloxacarbocyanine (DiOC2(3)), which, in the monomeric state, is normally green (488 --> 530 nm) fluorescent. In bacteria incubated with 30 microM dye, aggregate formation increases with the magnitude of the interior-negative membrane potential. Green fluorescence from stained bacteria predominantly reflects particle size, and is relatively independent of MP, whereas red fluorescence is highly dependent on both MP and size. The ratio of red to green fluorescence provides a measure of MP that is largely independent of cell size, with a low coefficient of variation (CV). Calibration with valinomycin and potassium demonstrates that the method is accurate over the range from -50 mV through -120 mV; it also accurately tracks reversible reductions in MP produced by incubation at 4 degrees C and washing in glucose-free medium. The ratiometric technique for MP estimation using DiOC2(3) is substantially more accurate and precise than those previously available, and may be useful in studies of bacterial physiology and in investigations of the effects of antibiotics and other agents on microorganisms.