Electrogenic transport of protons driven by the plasma membrane ATPase in membrane vesicles from radish : biochemical characterization

Mg:ATP-dependent H⁺ pumping has been studied in microsomal vesicles from 24-hour-old radish (Raphanus sativus L.) seedlings by monitoring both intravesicular acidification and the building up of an inside positive membrane potential difference (Δ ψ). ΔpH was measured as the decrease of absorbance of Acridine orange and Δ ψ as the shift of absorbance of bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol. Both Mg:ATP-dependent Δ pH and Δ ψ generation are completely inhibited by vanadate and insensitive to oligomycin; moreover, Δ pH generation is not inhibited by NO₃⁻. These findings indicate that this membrane preparation is virtually devoid of mitochondrial and tonoplast H⁺-ATPases. Both intravesicular acidification and Δ ψ generation are influenced by anions: Δ pH increases and Δ ψ decreases following the sequence SO₄²⁻, Cl⁻, Br⁻, NO₃⁻. ATP-dependent H⁺ pumping strictly requires Mg²⁺. It is very specific for ATP (apparent K_m 0.76 millimolar) compared to GTP, UTP, CTP, ITP. Δ pH generation is inhibited by CuSO₄ and diethylstilbestrol as well as vanadate. Δ pH generation is specificially stimulated by K⁺ (+ 80%) and to a lesser extent by Na⁺ and choline (+28% and +14%, respectively). The characteristics of H⁺ pumping in these microsomal vesicles closely resemble those described for the plasma membrane ATPase partially purified from several plant materials.     

A stopped-flow kinetic study of the interaction of potential-sensitive oxonol dyes with lipid vesicles

The interaction of the dyes oxonol V and oxonol VI with unilamellar dioleoylphosphatidylcholine vesicles was investigated using a fluorescence stopped-flow technique. On mixing with the vesicles, both dyes exhibit an increase in their fluorescence, which occurs in two phases. According to the dependence of the reciprocal relaxation time on vesicle concentration, the rapid phase appears to be due to a second-order binding of the dye to the lipid membrane, which is very close to being diffusion-controlled. The slow phase is almost independent of vesicle concentration, and it is suggested that this may be due to a change in dye conformation or position within the membrane, possibly diffusion across the membrane to the internal monolayer. The response times of the dyes to a rapid jump in the membrane potential has also been investigated. Oxonol VI was found to respond to the potential change in less than 1 s, whereas oxonol required several minutes. This has been attributed to lower mobility of oxonol V within the lipid membrane.     

Measurement of the Absolute Magnitude and Time Courses of Mitochondrial Membrane Potential in Primary and Clonal Pancreatic Beta-Cells

The aim of this study was to simplify, improve and validate quantitative measurement of the mitochondrial membrane potential (ΔψM) in pancreatic β-cells. This built on our previously introduced calculation of the absolute magnitude of ΔψM in intact cells, using time-lapse imaging of the non-quench mode fluorescence of tetramethylrhodamine methyl ester and a bis-oxonol plasma membrane potential (ΔψP) indicator. ΔψM is a central mediator of glucose-stimulated insulin secretion in pancreatic β-cells. ΔψM is at the crossroads of cellular energy production and demand, therefore precise assay of its magnitude is a valuable tool to study how these processes interplay in insulin secretion. Dispersed islet cell cultures allowed cell type-specific, single-cell observations of cell-to-cell heterogeneity of ΔψM and ΔψP. Glucose addition caused hyperpolarization of ΔψM and depolarization of ΔψP. The hyperpolarization was a monophasic step increase, even in cells where the ΔψP depolarization was biphasic. The biphasic response of ΔψP was associated with a larger hyperpolarization of ΔψM than the monophasic response. Analysis of the relationships between ΔψP and ΔψM revealed that primary dispersed β-cells responded to glucose heterogeneously, driven by variable activation of energy metabolism. Sensitivity analysis of the calibration was consistent with β-cells having substantial cell-to-cell variations in amounts of mitochondria, and this was predicted not to impair the accuracy of determinations of relative changes in ΔψM and ΔψP. Finally, we demonstrate a significant problem with using an alternative ΔψM probe, rhodamine 123. In glucose-stimulated and oligomycin-inhibited β-cells the principles of the rhodamine 123 assay were breached, resulting in misleading conclusions.     

Photo-Induction and Automated Quantification of Reversible Mitochondrial Permeability Transition Pore Opening in Primary Mouse Myotubes

Opening of the mitochondrial permeability transition pore (mPTP) is involved in various cellular processes including apoptosis induction. Two distinct states of mPTP opening have been identified allowing the transfer of molecules with a molecular weight <1500 Da or <300 Da. The latter state is considered to be reversible and suggested to play a role in normal cell physiology. Here we present a strategy combining live-cell imaging and computer-assisted image processing allowing spatial visualization and quantitative analysis of reversible mPTP openings (“ΔΨ flickering”) in primary mouse myotubes. The latter were stained with the photosensitive cation TMRM, which partitions between the cytosol and mitochondrial matrix as a function of mitochondrial membrane potential (ΔΨ). Controlled illumination of TMRM-stained primary mouse myotubes induced ΔΨ flickering in particular parts of the cell (“flickering domains”). A novel quantitative automated analysis was developed and validated to detect and quantify the frequency, size, and location of individual ΔΨ flickering events in myotubes.     

Dissipation of the Membrane Potential in Susceptible Corn Mitochondria by the Toxin of Helminthosporium maydis, Race T, and Toxin Analogs

We have tested directly the effect of Helminthosporium maydis T (Hmt) toxin and various analogs on the membrane potential formed in mitochondria isolated from a Texas (T) cytoplasmic male-sterile and a normal (N) corn. ATP, malate or succinate generated a membrane potential (negative inside) as monitored by the absorbance change of a cationic dye, safranine. The relative membrane potential (Δψ) could also be detected indirectly as ⁴⁵Ca²⁺ uptake. Hmt toxin added to T mitochondria dissipated the steady state Δψ similar to addition of a protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP). Toxin analogs (Cpd XIII: C₄₁H₆₈O₁₂ and Cpd IV: C₂₅H₄₄O₆), reduced native toxin (RT2C: C₄₁H₈₄O₁₃) and Pm toxin (band A: C₃₃H₆₀O₈, produced by the fungus, Phyllosticta maydis) were effective in dissipating Δψ and decreasing Ca²⁺ uptake with the following order: Pm (100) » HmT (23-30) > Cpd XIII (11-25) » RT2C (0-4−1.8) > Cpd IV (0.2−1.0). In contrast, the toxins and analogs had no effect on Δψ formed in N mitochondria. The striking similarities of the HmT toxin (band 1: C₄₁H₆₈O₁₃) and Cpd XIII on T mitochondrial activities provide strong evidence supporting the correctness of the polyketol structure assigned to the native toxin. Since the Δψ in energized mitochondria is caused mainly by the electrogenic extrusion of H⁺, the results support the idea that HmT toxin increases membrane permeability of T mitochondria to H⁺. The host specificity of the toxin suggests that an interaction with unique target site(s) on the inner mitochondrial membrane of T corn causes H⁺ leakage.     

Mechanism of potential-dependent light absorption changes of lipid bilayer membranes in the presence of cyanine and oxonol dyes

Summary The mechanism by which the light absorption of cyanine and oxonol dyes changes in response to changes in transmembrane electrical potential has been studied. Trains of membrane potential steps produce changes in the intensity of light passing through glycerylmonooleate (GMO) bilayer lipid membranes (BLM) in the presence of these dyes. The size of the signal-averaged absorbance change for one of the cyanine dyes diS-C₂-(5) is 10^–5. The response time for the absorbance change of all of the dyes was 10 sec. In order for an absorption signal to be observed, the concentration of dye on both sides of the membrane must be different. Since GMO bilayer membranes are permeable to the charged dyes that were studied, the dye concentration asymmetry necessary for the optical signal had to be maintained with a constant dc membrane potential, onto which the trains of potential steps were superimposed. The more hydrophobic dyes were the most permeant. Inclusion of cholesterol in the GMO bilayers decreased the permeance of the positively charged cyanine dyes, but increased the permeance of the negatively charged oxonol dyes. The magnitude and the size of the BLM absorbance change depended on the wavelength of illumination. Comparisons of the wavelength dependence of the BLM spectra with absorption difference spectra obtained with model membrane systems allow us to postulate a mechanism for a BLM absorbance change. For the cyanine and oxonol dyes, the data are consistent with an ON-OFF mechanism where a quantity of dye undergoes a rapid potential-dependent movement between a hydrocarbon-like binding site on the membrane and the aqueous salt solution near the membrane. For some dyes, which readily aggregate on the membrane, part of the absorbance change may possibly be explained by a potential dependent change in the state of aggregation of dye molecules localized on the membrane. Mechanisms involving a potential dependent change in the polarizability of the environment of membrane-localized dye molecules cannot be excluded, but seem unlikely.     

Rhomboid Protease PARL Mediates the Mitochondrial Membrane Potential Loss-induced Cleavage of PGAM5

Regulated intramembrane proteolysis is a widely conserved mechanism for controlling diverse biological processes. Considering that proteolysis is irreversible, it must be precisely regulated in a context-dependent manner. Here, we show that phosphoglycerate mutase 5 (PGAM5), a mitochondrial Ser/Thr protein phosphatase, is cleaved in its N-terminal transmembrane domain in response to mitochondrial membrane potential (ΔΨ_m) loss. This ΔΨ_m loss-dependent cleavage of PGAM5 was mediated by presenilin-associated rhomboid-like (PARL). PARL is a mitochondrial resident rhomboid serine protease and has recently been reported to mediate the cleavage of PINK1, a mitochondrial Ser/Thr protein kinase, in healthy mitochondria with intact ΔΨ_m. Intriguingly, we found that PARL dissociated from PINK1 and reciprocally associated with PGAM5 in response to ΔΨ_m loss. These results suggest that PARL mediates differential cleavage of PINK1 and PGAM5 depending on the health status of mitochondria. Our data provide a prototypical example of stress-dependent regulation of PARL-mediated regulated intramembrane proteolysis.     

Measuring the Induced Membrane Voltage with Di-8-ANEPPS

Placement of a cell into an external electric field causes a local charge redistribution inside and outside of the cell in the vicinity of the cell membrane, resulting in a voltage across the membrane. This voltage, termed the induced membrane voltage (also induced transmembrane voltage, or induced transmembrane potential difference) and denoted by ΔΦ, exists only as long as the external field is present. If the resting voltage is present on the membrane, the induced voltage superimposes (adds) onto it. By using one of the potentiometric fluorescent dyes, such as di-8-ANEPPS, it is possible to observe the variations of ΔΦ on the cell membrane and to measure its value noninvasively. di-8-ANEPPS becomes strongly fluorescent when bound to the lipid bilayer of the cell membrane, with the change of the fluorescence intensity proportional to the change of ΔΦ. This video shows the protocol for measuring ΔΦ using di-8-ANEPPS and also demonstrates the influence of cell shape on the amplitude and spatial distribution of ΔΦ.     

Osmotic adjustment in sorghum: I. Mechanisms of diurnal osmotic potential changes

Osmotic adjustment, defined as a lowering of osmotic potential (ψ_π) due to net solute accumulation in response to water stress, has been considered to be a beneficial drought tolerance mechanism in some crop species. The objective of this experiment was to determine the relative contribution of passive versus active mechanisms involved in diurnal ψ_π changes in sorghum (Sorghum bicolor L. Moench) leaf tissue in response to water stress. A single sorghum hybrid (cv AT×623 × RT×430) was grown in the field under variable water supplies. Water potential, ψ_π, and relative water content were measured diurnally on expanding and the uppermost fully expanded leaves before flowering and on fully expanded leaves during the grain-filling period. Diurnal changes in total osmotic potential (Δψ_π) in response to water stress was 1.1 megapascals before flowering and 1.4 megapascals during grain filling in comparison with 0.53 megapascal under well-watered conditions. Under water-stressed conditions, passive concentration of solutes associated with dehydration accounted for 50% (0.55 megapascal) of the diurnal Δψ_π before flowering and 47% (0.66 megapascal) of the change during grain filling. Net solute accumulation accounted for 42% (0.46 megapascal) of the diurnal Δψ_π before flowering and 45% (0.63 megapascal) of the change during grain filling in water-stressed leaves. The relative contribution of changes in nonosmotic volume (decreased turgid weight/dry weight) to diurnal Δψ_π was less than 8% at either growth stages. Water stress did not affect leaf tissue elasticity or partitioning of water between the symplasm and apoplasm.     

The Ca-Transport ATPase of Plant Plasma Membrane Catalyzes a nH/Ca Exchange

Microsomal vesicles from 24-hour-old radish (Raphanus sativus L.) seedlings accumulate Ca²⁺ upon addition of MgATP. MgATP-dependent Ca²⁺ uptake co-migrates with the plasma membrane H⁺-ATPase on a sucrose gradient. Ca²⁺ uptake is insensitive to oligomycin, inhibited by vanadate (IC₅₀ 40 micromolar) and erythrosin B (IC₅₀ 0.2 micromolar) and displays a pH optimum between pH 6.6 and 6.9. MgATP-dependent Ca²⁺ uptake is insensitive to protonophores. These results indicate that Ca²⁺ transport in these microsomal vesicles is catalyzed by a Mg²⁺-dependent ATPase localized on the plasma membrane. Ca²⁺ strongly reduces ΔpH generation by the plasma membrane H⁺-ATPase and increases MgATP-dependent membrane potential difference (Δψ) generation. These effects of Ca²⁺ on ΔpH and Δψ generation are drastically reduced by micromolar erythrosin B, indicating that they are primarily a consequence of Ca²⁺ uptake into plasma membrane vesicles. The Ca²⁺-induced increase of Δψ is collapsed by permeant anions, which do not affect Ca²⁺-induced decrease of ΔpH generation by the plasma membrane H⁺-ATPase. The rate of decay of MgATP-dependent ΔpH, upon inhibition of the plasma membrane H⁺-ATPase, is accelerated by MgATP-dependent Ca²⁺ uptake, indicating that the decrease of ΔpH generation induced by Ca²⁺ reflects the efflux of H⁺ coupled to Ca²⁺ uptake into plasma membrane vesicles. It is therefore proposed that Ca²⁺ transport at the plasma membrane is mediated by a Mg²⁺-dependent ATPase which catalyzes a nH⁺/Ca²⁺ exchange.     

Use of oxonol V as a probe of membrane potential in proteoliposomes containing cytochrome oxidase in the submitochondrial orientation

Absorbance changes in the anionic dye bis[3-phenyl-5-oxoisoxazol-4-yl]pentamethineoxonol (oxonol V) can be used to monitor the membrane potential of liposomes and cytochrome c containing cytochrome oxidase proteoliposomes (c-loaded COV). Diffusion potentials (positive inside the vesicles) cause an increase in the dye extinction, with a maximum at 640 nm. A similar increase is seen upon energization of internally facing cytochrome oxidase molecules in c-loaded COV. Both "passive" and "active" responses are only seen when the dye is fully bound to the vesicle membrane. Calibration curves using potassium or n-butyltriphenylphosphonium ion (BTPP+) diffusion potentials are linear up to 100 mV and pass through the origin. Diffusion potentials (positive inside) also cause an increase and red shift in the oxonol V fluorescence emission spectrum. However, potentials of the same sign induced by cytochrome oxidase turnover induce a large fluorescence quenching in c-loaded COV. A similar anomaly has been observed with submitochondrial particles [Smith, J. C., Russ, P., Cooperman, B. S., & Chance, B. (1976) Biochemistry 15, 5094-5105]. A model is proposed consistent with these responses. It is suggested that the dye molecules move further into the membrane phase upon energization, causing the absorbance increase. In the presence of active enzyme, anionic dye molecules are attracted to a positive dipole on each enzyme molecule, causing self-quenching of the fluorescence.     

Essential Role of TID1 in Maintaining Mitochondrial Membrane Potential Homogeneity and Mitochondrial DNA Integrity

The tumorous imaginal disc 1 (TID1) protein localizes mainly to the mitochondrial compartment, wherein its function remains largely unknown. Here we report that TID1 regulates the steady-state homogeneity of the mitochondrial membrane potential (Δψ) and maintains the integrity of mitochondrial DNA (mtDNA). Silencing of TID1 with RNA interference leads to changes in the distribution of Δψ along the mitochondrial network, characterized by an increase in Δψ in focal regions. This effect can be rescued by ectopic expression of a TID1 construct with an intact J domain. Chronic treatment with a low dose of oligomycin, an inhibitor of F₁F_o ATP synthase, decreases the cellular ATP content and phenocopies TID1 loss of function, indicating a connection between the disruption of mitochondrial bioenergetics and hyperpolarization. Prolonged silencing of TID1 or low-dose oligomycin treatment leads to the loss of mtDNA and the consequent inhibition of oxygen consumption. Biochemical and colocalization data indicate that complex I aggregation underlies the focal accumulation of Δψ in TID1-silenced cells. Given that TID1 is proposed to function as a cochaperone, these data show that TID1 prevents complex I aggregation and support the existence of a TID1-mediated stress response to ATP synthase inhibition.     

Electrostatic Changes in Lycopersicon esculentum Root Plasma Membrane Resulting from Salt Stress

Salinity-induced alterations in tomato (Lypersicon esculentum Mill. cv Heinz 1350) root plasma membrane properties were studied and characterized using a membrane vesicle system. Equivalent rates of MgATP-dependent H⁺-transport activity were measured by quinacrine fluorescence (ΔpH) in plasma membrane vesicles isolated from control or salt-stressed (75 millimolar salt) tomato roots. However, when bis-[3-phenyl-5-oxoisoxazol-4-yl] pentamethine was used to measure MgATP-dependent membrane potential (ΔΨ) formation, salt-stressed vesicles displayed a 50% greater initial quench rate and a 30% greater steady state quench than control vesicles. This differential probe response suggested a difference in surface properties between control and salt-stressed membranes. Fluorescence titration of vesicles with the surface potential probe, 8-anilino-1-napthalenesulphonic acid (ANS) provided dissociation constants (K_d) of 120 and 76 micromolar for dye binding to control and salt-stressed vesicles, respectively. Membrane surface potentials (Ψ_o) of−26.0 and −13.7 millivolts were calculated for control and salt-stressed membrane vesicles from the measured K_d values and the calculated intrinsic affinity constant, K_i. The concentration of cations and anions at the surface of control and salt-stressed membranes was estimated using Ψ_o values and the Boltzmann equation. The observed difference in membrane surface electrostatic properties was consistent with the measured differences in K⁺-stimulated kinetics of ATPase activity between control and salt-stressed vesicles and by the differential ability of Cl⁻ ions to stimulate H⁺-transport activity. Salinity-induced changes in plasma membrane electrostatic properties may influence ion transport across the plasma membrane.     

Lycium barbarum Polysaccharides Protect Human Lens Epithelial Cells against Oxidative Stress–Induced Apoptosis and Senescence

Objectives

We aimed to investigate the protective effect of Lycium barbarum polysaccharides (LBPs) against oxidative stress–induced apoptosis and senescence in human lens epithelial cells.

Methods

To study apoptosis, SRA01/04 cells, a human lens epithelial cell lines, were exposed to 200 µM hydrogen peroxide (H₂O₂) for 24 h with or without pretreatment with LBPs. Cell viability was measured using a Cell Counting Kit-8 (CCK-8) assay. Cell apoptosis, intracellular reactive oxygen species (ROS), and the loss of mitochondria membrane potential (Δψm) were detected by flow cytometric analyses. Expression levels of Bcl-2 and Bax proteins were measured by western blot analysis. The levels of malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH) were quantized using commercial enzymatic kits according to the manufacturer''s instructions. To study senescence, SRA01/04 cells were pre-incubated with LBPs and all cells were then exposed to 100 µM H₂O₂ for 96 h. Cellular senescence was assessed by morphologic examination and senescence-associated β-galactosidase (SA-β-gal) staining.

Results

LBPs significantly reduced H₂O₂-induced cell apoptosis, the generation of ROS, the loss of Δψm, and the levels of MDA. LBPs also inhibited H₂O₂-induced downregulated Bcl-2 and upregulated Bax proteins and increased the levels of SOD and GSH enzyme activity. Moreover, LBPs significantly attenuated H₂O₂-induced cellular senescence.

Conclusions

These findings suggested that LBPs protect human lens epithelial cells from H₂O₂-induced apoptosis by modulating the generation of ROS, loss of Δψm, Bcl-2 family, and antioxidant enzyme activity and attenuating cellular senescence.     

High Efficacy but Low Potency of δ-Opioid Receptor-G Protein Coupling in Brij-58-Treated,Low-Density Plasma Membrane Fragments

Principal Findings

HEK293 cells stably expressing PTX-insensitive δ-opioid receptor-Gi1α (C³⁵¹I) fusion protein were homogenized, treated with low concentrations of non-ionic detergent Brij-58 at 0°C and fractionated by flotation in sucrose density gradient. In optimum range of detergent concentrations (0.025–0.05% w/v), Brij-58-treated, low-density membranes exhibited 2-3-fold higher efficacy of DADLE-stimulated, high-affinity [³²P]GTPase and [³⁵S]GTPγS binding than membranes of the same density prepared in the absence of detergent. The potency of agonist DADLE response was significantly decreased. At high detergent concentrations (>0.1%), the functional coupling between δ-opioid receptors and G proteins was completely diminished. The same detergent effects were measured in plasma membranes isolated from PTX-treated cells. Therefore, the effect of Brij-58 on δ-opioid receptor-G protein coupling was not restricted to the covalently bound G_i1α within δ-opioid receptor-G_i1α fusion protein, but it was also valid for PTX-sensitive G proteins of G_i/G_o family endogenously expressed in HEK293 cells. Characterization of the direct effect of Brij-58 on the hydrophobic interior of isolated plasma membranes by steady-state anisotropy of diphenylhexatriene (DPH) fluorescence indicated a marked increase of membrane fluidity. The time-resolved analysis of decay of DPH fluorescence by the “wobble in cone” model of DPH motion in the membrane indicated that the exposure to the increasing concentrations of Brij-58 led to a decreased order and higher motional freedom of the dye.

Summary

Limited perturbation of plasma membrane integrity by low concentrations of non-ionic detergent Brij-58 results in alteration of δ-OR-G protein coupling. Maximum G protein-response to agonist stimulation (efficacy) is increased; affinity of response (potency) is decreased. The total degradation plasma membrane structure at high detergent concentrations results in diminution of functional coupling between δ-opioid receptors and G proteins.     

Bacterial Porin Disrupts Mitochondrial Membrane Potential and Sensitizes Host Cells to Apoptosis

The bacterial PorB porin, an ATP-binding β-barrel protein of pathogenic Neisseria gonorrhoeae, triggers host cell apoptosis by an unknown mechanism. PorB is targeted to and imported by host cell mitochondria, causing the breakdown of the mitochondrial membrane potential (ΔΨ_m). Here, we show that PorB induces the condensation of the mitochondrial matrix and the loss of cristae structures, sensitizing cells to the induction of apoptosis via signaling pathways activated by BH3-only proteins. PorB is imported into mitochondria through the general translocase TOM but, unexpectedly, is not recognized by the SAM sorting machinery, usually required for the assembly of β-barrel proteins in the mitochondrial outer membrane. PorB integrates into the mitochondrial inner membrane, leading to the breakdown of ΔΨ_m. The PorB channel is regulated by nucleotides and an isogenic PorB mutant defective in ATP-binding failed to induce ΔΨ_m loss and apoptosis, demonstrating that dissipation of ΔΨ_m is a requirement for cell death caused by neisserial infection.     

Dilute Solution Approximation and Generalization of the Reflection Coefficient Method of Describing Volume and Solute Flows

Kedem and Katchalsky introduced an approximation for dilute solutions which requires that the quantity (Δπ/Δπ_i)ø_i be much less than one. Zelman attempted to generalize the reflection coefficient concept to apply to solutions of multiple solutes, both penetrable and impenetrable, of concentrations sufficiently high for the approximation not to work. By simple algebraic manipulation, Zelman introduced a pair of new reflection coefficients, and a third new parameter γ which he misleadingly calls the “deviation from the dilute solution approximation.” It is shown here that the original Kedem-Katchalsky form for the flow equations can be preserved in such a way that no new coefficients need be introduced and an explicit statement of the effect of the dilute solution approximation can be made. There is an option of using a new set of conjugate driving forces for the solute flows or, alternatively, incorporating the nondilute solution correction in the coefficients in a clear way.     

Components of Sodium and Chloride Flux Across Toad Bladder

The effect of transepithelial potential difference (ψ) on Na and Cl flux across toad bladder was assessed by measuring isotopic flux between identical media at various values of ψ. The contribution of edge damage to ionic permeability was eliminated, resulting in relatively high spontaneous ψ (-97 ±4 mv) and low electrical conductance g. Bidirectional Na fluxes were measured simultaneously. Unidirectional Cl fluxes were measured in paired hemibladders at ψ = 0 mv or -97 mv. Net Na flux J_Na, at ψ = 0 mv, was slightly less than short-circuit current (SCC). At ψ = -97 mv, J_Na averaged 17% of SCC, and was sometimes zero. ΔJ_Na/Δψ (= g₊) averaged 60% of g between -97 mv and +75 mv; at -150 mv, g₊ fell, indicating rectification. Analysis of unidirectional Na fluxes indicates low passive conductance (1.5 μmho/mg wet weight), a bidirectional, electrically neutral flux of approximately 0.13 μa/mg, and relatively large conductance of the active transport path at ψ ≥ -97 mv. The absence of appreciable transstimulation of serosal (S)-to-mucosal (M) Na flux (in response to increasing mucosal Na concentration) indicates that the electrically neutral flux is not exchange diffusion in the usual sense. Analysis of Cl fluxes indicates similar values for passive conductance and neutral flux, suggesting linked neutral flux of Na and Cl. Either the electromotive force of the Na pump E, its conductance g_a, or both are strong functions of ψ. The product of these two quantities, Eg_a, is a measure of the “transport capacity” at any given value of ψ, independent of the direct effect of ψ on J_Na through the pump path. Eg_a varies with ψ. Hence estimation of the net Na flux or current at any one value of ψ, including ψ = 0, fails to reveal the maximal transport capacity of the pump, its resting electromotive force (when J_Na = 0 through the pump), or the dependence of transport capacity on potential.     

Potentiation of Ligand Binding through Cooperative Effects in Monoamine Oxidase B

Crystallographic and biochemical studies have been employed to identify the binding site and mechanism for potentiation of imidazoline binding in human monoamine oxidase B (MAO B). 2-(2-Benzofuranyl)-2-imidazoline (2-BFI) inhibits recombinant human MAO B with a K_i of 8.3 ± 0.6 μm, whereas tranylcypromine-inhibited MAO B binds 2-BFI with a K_d of 9 ± 2 nm, representing an increase in binding energy Δ(ΔG) of −3.9 kcal/mol. Crystal structures show the imidazoline ligand bound in a site that is distinct from the substrate-binding cavity. Contributions to account for the increase in binding affinity upon tranylcypromine inhibition include a conformational change in the side chain of Gln²⁰⁶ and a “closed conformation” of the side chain of Ile¹⁹⁹, forming a hydrophobic “sandwich” with the side chain of Ile³¹⁶ on each face of the benzofuran ring of 2-BFI. Data with the I199A mutant of human MAO B and failure to observe a similar binding potentiation with rat MAO B, where Ile³¹⁶ is replaced with a Val residue, support an allosteric mechanism where the increased binding affinity of 2-BFI results from a cooperative increase in H-bond strength through formation of a more hydrophobic milieu. These insights should prove valuable in the design of high affinity and specific reversible MAO B inhibitors.