Energetics of Sodium Transport in Frog Skin : II. The effects of electrical potential on oxygen consumption

Studies were made of the dependence of the rate of oxygen consumption, J_r, on the electrical potential difference, Δψ, across the frog skin. After the abolition of sodium transport by ouabain the basal oxygen consumption was independent of Δψ. In fresh skins J_r was a linear function of Δψ over a range of at least ±70 mv. Treatment with aldosterone stimulated the short-circuit current, I_o, and the associated rate of oxygen consumption, J_ro, and increased their stability; linearity was then demonstrable over a range of ±160 mv. Brief perturbations of Δψ (±30–200 mv) did not alter subsequent values of I_o. Perturbations for 10 min or more produced a "memory" effect both with and without aldosterone: accelerating sodium transport by negative clamping lowered the subsequent value of I_o; positive clamping induced the opposite effect. Changes in J_ro were more readily detectable in the presence of aldosterone; these were in the same direction as the changes in I_o. The linearity of J_r in Δψ indicates the validity of analysis in terms of linear nonequilibrium thermodynamics—brief perturbations of Δψ appear to produce no significant effect on either the phenomenological coefficients or the free energy of the metabolic driving reaction. Hence it is possible to evaluate this free energy.     

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.     

Proton motive force during growth of Streptococcus lactis cells

Experiments with the aerotolerant anaerobe Streptococcus lactis provide the opportunity for determining the proton motive force (Δp) in dividing cells. The two components of Δp, ΔΨ (the transmembrane potential) and ΔpH (the chemical gradient of H⁺), were determined by the accumulation of radiolabeled tetraphenylphosphonium (TPP⁺) and benzoate ions. The ΔΨ was calibrated with the K⁺ diffusion potential in starved, valinomycin-treated cells. With resting, glycolyzing cells, the Δp was measured also by the accumulation of the non-metabolizable sugar thiomethyl-β-galactoside (TMG). In resting cells the Δp, calculated either by adding ΔΨ and ZΔpH or from the levels of TMG, was relatively constant between pH 5 to 7, decreasing from 160 to 150 mV and decreasing further to 100 mV at pH 8.0. With the TPP⁺ probe for ΔΨ, we confirmed our previous finding that the K⁺ ions dissipate ΔΨ and increase ΔpH, whereas Na⁺ ions have little effect on ΔΨ and no effect on ΔpH. [³H]TPP⁺ and [¹⁴C]benzoate were added during exponential phase to S. lactis cells growing at pH 5 to 7 at 28°C in a defined medium with glucose as energy source. As with resting cells, the ΔpH and ΔΨ were dependent on the pH of the medium. At pH 5.1, the ΔpH was equivalent to 60 mV (alkaline inside) and decreased to 25 mV at pH 6.8. The ΔΨ increased from 83 mV (negative inside) at pH 5.1 to 108 mV at pH 6.8. The Δp, therefore, was fairly constant between pH 5 and 7, decreasing from 143 to 133 mV. The values for Δp in growing cells, just as in resting cells, are consistent with a system in which the net efflux of H⁺ ions is effected by a membrane-bound adenosine triphosphatase and glycolytically generated adenosine triphosphate. The data suggest that in both growing and resting cells the pH of the medium and its K⁺ concentration are the two principal factors that determine the relative contribution of ΔpH and ΔΨ to the proton motive force.     

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.     

Sodium Efflux and Potential Differences across the Irrigated Gill of Sea Water-Adapted Rainbow Trout (Salmo gairdneri)

Sodium extrusion (J_out^Na) was measured across the gills of rainbow trout, Salmo gairdneri, adapted to sea water (SW) using a gill-irrigation system of small volume. The potential difference (TEP) was also measured under similar conditions. J_out^Na was usually between 100–250 µeq (100 g)^–1 h^–1, about an order of magnitude faster than in fresh water (FW)-adapted trout, but slower than has been reported for any other marine teleost. The TEP was between 10–11 mV, body fluids positive to SW. When the external medium was changed from SW to FW J_out^Na was reduced to about 25 % of the initial value, and the TEP was reduced by 40–50 mV (i.e. body fluids negative by 30–40 mV). Addition of either Na⁺ or K⁺ in SW concentrations reversed the changes; J_out^Na increased and the gill repolarized. The electrical behavior and sodium efflux in irrigated trout gill is qualitatively the same as has been reported for unanaesthetized, free-swimming fish of other species. Thus, the irrigated gill provides an adequate model for studying the mechanism of sodium extrusion in marine teleosts.     

Proton Electrochemical Gradients in Washed Cells of Nitrosomonas europaea and Nitrobacter agilis

The components of the proton motive force (Δp), namely, membrane potential (Δψ) and transmembrane pH gradient (ΔpH), were determined in the nitrifying bacteria Nitrosomonas europaea and Nitrobacter agilis. In these bacteria both Δψ and ΔpH were dependent on external pH. Thus at pH 8.0, Nitrosomonas europaea and Nitrobacter agilis had Δψ values of 173 mV and 125 mV (inside negative), respectively, as determined by the distribution of the lipophilic cation [³H]tetraphenyl phosphonium. Intracellular pH was determined by the distribution of two weak acids, ¹⁴C-benzoic and ¹⁴C-acetyl salicylic, and the weak base [¹⁴C]methylamine. Nitrosomonas europaea accumulated ¹⁴C-benzoic acid and ¹⁴C-acetyl salicylic acid when the external pH was below 7.0 and [¹⁴C]methylamine at alkaline pH. Similarly, Nitrobacter agilis accumulated the two weak acids below an external pH of about 7.5 and [¹⁴C]methylamine above this pH. As these bacteria grow best between pH 7.5 and 8.0, they do not appear to have a ΔpH (inside alkaline). Thus, above pH 7.0 for Nitrosomonas europaea and pH 7.5 for Nitrobacter agilis, Δψ only contributed to Δp. In Nitrosomonas europaea the total Δp remained almost constant (145 to 135 mV) when the external pH was varied from 6 to 8.5. In Nitrobacter agilis, Δp decreased from 178 mV (inside negative) at pH 6.0 to 95 mV at pH 8.5. Intracellular pH in Nitrosomonas europaea varied from 6.3 at an external pH of 6.0 to 7.8 at external pH 8.5. In Nitrobacter agilis, however, intracellular pH was relatively constant (7.3 to 7.8) over an external pH range of 6 to 8.5. In Nitrosomonas europaea, Δp and its components (Δψ and ΔpH) remained constant in cells at various stages of growth, so that the metabolic state of cells did not affect Δp. Such an experiment was not possible with Nitrobacter agilis because of low cell yields. The effects of protonophores and ATPase inhibitors on ΔpH and Δψ in the two nitrifying bacteria are considered.     

Sodium and Calcium Inward Currents in Freshly Dissociated Smooth Myocytes of Rat Uterus

Freshly dissociated myocytes from nonpregnant, pregnant, and postpartum rat uteri have been studied with the tight-seal patch-clamp method. The inward current contains both I_Na and I_Ca that are vastly different from those in tissue-cultured material. I_Na is abolished by Na⁺-free medium and by 1 μM tetrodotoxin. It first appears at ∼−40 mV, reaches maximum at 0 mV, and reverses at 84 mV. It activates with a voltage-dependent τ of 0.2 ms at 20 mV, and inactivates as a single exponential with a τ of 0.4 ms. Na⁺ conductance is half activated at −21.5 mV, and half inactivated at −59 mV. I_Na reactivates with a τ of 20 ms. I_Ca is abolished by Ca²⁺-free medium, Co²⁺ (5 mM), or nisoldipine (2 μM), and enhanced in 30 mM Ca²⁺, Ba²⁺, or BAY-K 8644. It first appears at ∼−30 mV and reaches maximum at +10 mV. It activates with a voltage-dependent τ of 1.5 ms at 20 mV, and inactivates in two exponential phases, with τ''s of 33 and 133 ms. Ca²⁺ conductance is half activated at −7.4 mV, and half inactivated at −34 mV. I_Ca reactivates with τ''s of 27 and 374 ms. I_Na and I_Ca are seen in myocytes from nonpregnant estrus uteri and throughout pregnancy, exhibiting complex changes. The ratio of densities of peak I_Na/I_Ca changes from 0.5 in the nonpregnant state to 1.6 at term. The enhanced role of I_Na, with faster kinetics, allows more frequent repetitive spike discharges to facilitate simultaneous excitation of the parturient uterus. In postpartum, both currents decrease markedly, with I_Na vanishing from most myocytes. Estrogen-enhanced genomic influences may account for the emergence of I_Na, and increased densities of I_Na and I_Ca as pregnancy progresses. Other influences may regulate varied channel expression at different stages of pregnancy.     

Single-channel analysis of the anion channel-forming protein from the plant pathogenic bacterium Clavibacter michiganense ssp. nebraskense

The anion channel protein from Clavibacter michiganense ssp. nebraskense (Schürholz, Th. et al. 1991, J. Membrane Biol. 123: 1-8) was analyzed at different concentrations of KCl and KF. At 0.8 M KCl the conductance G(V_m) increases exponentially from 21 pS at 50 mV up to 53 pS at V_m = 200 mV, 20°C. The concentration dependence of G(V_m) corresponds to a Michaelis-Menten type saturation function at all membrane voltage values applied (0-200 mV). The anion concentration K_0.5, where G(V_m) has its half-maximum value, increases from 0.12 M at 50 mV to 0.24 M at 175 mV for channels in a soybean phospholipid bilayer. The voltage dependence of the single channel conductance, which is different for charged and neutral lipid bilayers, can be described either by a two-state flicker (2SF) model and the Nernst-Planck continuum theory, or by a two barrier, one-site (2B1S) model with asymmetric barriers. The increase in the number of open channels after a voltage jump from 50 mV to 150 mV has a time constant of 0.8 s. The changes of the single-channel conductance are much faster (<1 ms). The electric part of the gating process is characterized by the (reversible) molar electrical work ΔG^θ_el = ρZ_gFV_m ≈ -1.3 RT, which corresponds to the movement of one charge of the gating charge number |Z_g| = 1 across the fraction ρ = ΔV_m/V_m = 0.15 of the membrane voltage V_m = 200 mV. Unlike with chloride, the single channel conductance of fluoride has a maximum at about 150 mV in the presence of the buffer PIPES (≥5 mM, pH 6.8) with K_0.5 ≈ 1 M. It is shown that the decrease in conductance is due to a blocking of the channel by the PIPES anion. In summary, the results indicate that the anion transport by the Clavibacter anion channel (CAC) does not require a voltage dependent conformation change of the CAC.     

Studies of Root Function in Zea mays: III. Xylem Sap Composition at Maximum Root Pressure Provides Evidence of Active Transport into the Xylem and a Measurement of the Reflection Coefficient of the Root

The cut ends of excised Zea mays roots were sealed to a pressure transducer and their root pressures recorded. These rose approximately hyperbolically to a maximum value of 4.21 ± 0.34 bar after 30 to 40 minutes. Xylem exudate could not be collected at this pressure since the flow rate was zero. Samples of exudate were collected at lower applied pressures (ΔP), however, and Δπ, the osmotic pressure difference between them and the solution bathing the root, was measured by freezing point depression. A plot of ΔP/Δπ against J_v/Δπ, where J_v is the volume flux, proved to be a straight line whose intercept, equal to σ, the reflection coefficient, was 0.853 ± 0.016. The maximum xylem concentrations of various chemical species were found by a similar extrapolative method and compared with those in the cell sap. This indicated that (a) Ca²⁺, Mg²⁺, NO₃²⁻, SO₄²⁻, and most amino acids move from the cells to the xylem down an electrochemical potential gradient; (b) relative to these ions H⁺, NH₄⁺, glutamine and asparagine are actively transported into the xylem; and (c) H₂PO₄⁻, and K⁺ are actively retained in the symplasm.     

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.     

Electrophysiological Characterization of the Rat Epithelial Na+ Channel (rENaC) Expressed in MDCK Cells : Effects of Na+ and Ca2+

The epithelial Na⁺ channel (ENaC), composed of three subunits (α, β, and γ), is expressed in several epithelia and plays a critical role in salt and water balance and in the regulation of blood pressure. Little is known, however, about the electrophysiological properties of this cloned channel when expressed in epithelial cells. Using whole-cell and single channel current recording techniques, we have now characterized the rat αβγENaC (rENaC) stably transfected and expressed in Madin-Darby canine kidney (MDCK) cells. Under whole-cell patch-clamp configuration, the αβγrENaC-expressing MDCK cells exhibited greater whole cell Na⁺ current at −143 mV (−1,466.2 ± 297.5 pA) than did untransfected cells (−47.6 ± 10.7 pA). This conductance was completely and reversibly inhibited by 10 μM amiloride, with a Ki of 20 nM at a membrane potential of −103 mV; the amiloride inhibition was slightly voltage dependent. Amiloride-sensitive whole-cell current of MDCK cells expressing αβ or αγ subunits alone was −115.2 ± 41.4 pA and −52.1 ± 24.5 pA at −143 mV, respectively, similar to the whole-cell Na⁺ current of untransfected cells. Relaxation analysis of the amiloride-sensitive current after voltage steps suggested that the channels were activated by membrane hyperpolarization. Ion selectivity sequence of the Na⁺ conductance was Li⁺ > Na⁺ >> K⁺ = N-methyl-d-glucamine⁺ (NMDG⁺). Using excised outside-out patches, amiloride-sensitive single channel conductance, likely responsible for the macroscopic Na⁺ channel current, was found to be ∼5 and 8 pS when Na⁺ and Li⁺ were used as a charge carrier, respectively. K⁺ conductance through the channel was undetectable. The channel activity, defined as a product of the number of active channel (n) and open probability (P _o), was increased by membrane hyperpolarization. Both whole-cell Na⁺ current and conductance were saturated with increased extracellular Na⁺ concentrations, which likely resulted from saturation of the single channel conductance. The channel activity (nP _o) was significantly decreased when cytosolic Na⁺ concentration was increased from 0 to 50 mM in inside-out patches. Whole-cell Na⁺ conductance (with Li⁺ as a charge carrier) was inhibited by the addition of ionomycin (1 μM) and Ca²⁺ (1 mM) to the bath. Dialysis of the cells with a pipette solution containing 1 μM Ca²⁺ caused a biphasic inhibition, with time constants of 1.7 ± 0.3 min (n = 3) and 128.4 ± 33.4 min (n = 3). An increase in cytosolic Ca²⁺ concentration from <1 nM to 1 μM was accompanied by a decrease in channel activity. Increasing cytosolic Ca²⁺ to 10 μM exhibited a pronounced inhibitory effect. Single channel conductance, however, was unchanged by increasing free Ca²⁺ concentrations from <1 nM to 10 μM. Collectively, these results provide the first characterization of rENaC heterologously expressed in a mammalian epithelial cell line, and provide evidence for channel regulation by cytosolic Na⁺ and Ca²⁺.     

Mechanisms of Action of Carvacrol on the Food-Borne Pathogen Bacillus cereus

Carvacrol, a naturally occurring compound mainly present in the essential oil fraction of oregano and thyme, was studied for its effect on bioenergetic parameters of vegetative cells of the food-borne pathogen Bacillus cereus. Incubation for 30 min in the presence of 1 to 3 mM carvacrol reduced the viable cell numbers exponentially. Carvacrol (2 mM) significantly depleted the intracellular ATP pool to values close to 0 within 7 min. No proportional increase of the extracellular ATP pool was observed. Depletion of the internal ATP pool was associated with a change of the membrane potential (Δψ). At concentrations of 0.01 mM carvacrol and above, a significant reduction of Δψ was observed, leading to full dissipation of Δψ at concentrations of 0.15 mM and higher. Finally, an increase of the permeability of the cytoplasmic membrane for protons and potassium ions was observed (at 0.25 and 1 mM carvacrol, respectively). From this study, it could be concluded that carvacrol interacts with the membranes of B. cereus by changing its permeability for cations like H⁺ and K⁺. The dissipation of ion gradients leads to impairment of essential processes in the cell and finally to cell death.     

Role of Listeria monocytogenes σB in Survival of Lethal Acidic Conditions and in the Acquired Acid Tolerance Response

The food-borne pathogen Listeria monocytogenes can acquire enhanced resistance to lethal acid conditions through multiple mechanisms. We investigated contributions of the stress-responsive alternative sigma factor, σ^B, which is encoded by sigB, to growth phase-dependent acid resistance (AR) and to the adaptive acid tolerance response in L. monocytogenes. At various points throughout growth, we compared the relative survival of L. monocytogenes wild-type and ΔsigB strains that had been exposed to either brain heart infusion (pH 2.5) or synthetic gastric fluid (pH 2.5) with and without prior acid adaptation. Under these conditions, survival of the ΔsigB strain was consistently lower than that of the wild-type strain throughout all phases of growth, ranging from 4 orders of magnitude less in mid-log phase to 2 orders of magnitude less in stationary phase. Survival of both ΔsigB and wild-type L. monocytogenes strains increased by 6 orders of magnitude upon entry into stationary phase, demonstrating that the L. monocytogenes growth phase-dependent AR mechanism is σ^B independent. σ^B-mediated contributions to acquired acid tolerance appear to be greatest in early logarithmic growth. Loss of a functional σ^B reduced the survival of L. monocytogenes at pH 2.5 to a greater extent in the presence of organic acid (100 mM acetic acid) than in the presence of inorganic acid alone (HCl), suggesting that L. monocytogenes protection against organic and inorganic acid may be mediated through different mechanisms. σ^B does not appear to contribute to pH_i homeostasis through regulation of net proton movement across the cell membrane or by regulation of pH_i buffering by the GAD system under the conditions examined in this study. In summary, a functional σ^B protein is necessary for full resistance of L. monocytogenes to lethal acid treatments.     

On the Mechanism of Sodium Extrusion across the Irrigated Gill of Sea Water-Adapted Rainbow Trout (Salmo gairdneri)

Sodium efflux (J_out^Na) across the irrigated trout gill was rapid in sea water (SW), but only about 25 % as large in fresh water (FW). The difference correlated with a change in the potential difference across the gill (TEP). The latter was about +10 mV (blood positive) in SW, but –40 mV in FW. Both flux and electrical data indicated that gills in this fish are permeable to a variety of cations including Na⁺, K⁺, Mg²⁺, choline, and Tris. They are less permeable to anions; P_Na:P_K:P_Cl was estimated to be 1:10:0.3, and P_Cl > P_gluconate. The TEP was shown to be a diffusion potential determined by these permeabilities and the extant ionic gradients in SW, FW as well as in other media. J_out^Na appeared to be diffusive in all of the experiments undertaken. Exchange diffusion need not be posited, and the question of whether there is an active component remains open.     

Effect of ENaC Modulators on Rat Neural Responses to NaCl

The effects of small molecule ENaC activators N,N,N-trimethyl-2-((4-methyl-2-((4-methyl-1H-indol-3-yl)thio)pentanoyl)oxy)ethanaminium iodide (Compound 1) and N-(2-hydroxyethyl)-4-methyl-2-((4-methyl-1H-indol-3-yl)thio)pentanamide (Compound 2), were tested on the benzamil (Bz)-sensitive NaCl chorda tympani (CT) taste nerve response under open-circuit conditions and under ±60 mV applied lingual voltage-clamp, and compared with the effects of known physiological activators (8-CPT-cAMP, BAPTA-AM, and alkaline pH), and an inhibitor (ionomycin+Ca²⁺) of ENaC. The NaCl CT response was enhanced at −60 mV and suppressed at +60 mV. In every case the CT response (r) versus voltage (V) curve was linear. All ENaC activators increased the open-circuit response (r_o) and the voltage sensitivity (κ, negative of the slope of the r versus V curve) and ionomycin+Ca²⁺ decreased r_o and κ to zero. Compound 1 and Compound 2 expressed a sigmoidal-saturating function of concentration (0.25–1 mM) with a half-maximal response concentration (k) of 0.49 and 1.05 mM, respectively. Following treatment with 1 mM Compound 1, 8-CPT-cAMP, BAPTA-AM and pH 10.3, the Bz-sensitive NaCl CT response to 100 mM NaCl was enhanced and was equivalent to the Bz-sensitive CT response to 300 mM NaCl. Plots of κ versus r_o in the absence and presence of the activators or the inhibitor were linear, suggesting that changes in the affinity of Na⁺ for ENaC under different conditions are fully compensated by changes in the apical membrane potential difference, and that the observed changes in the Bz-sensitive NaCl CT response arise exclusively from changes in the maximum CT response (r_m). The results further suggest that the agonists enhance and ionomycin+Ca²⁺ decreases ENaC function by increasing or decreasing the rate of release of Na⁺ from its ENaC binding site to the receptor cell cytosol, respectively. Irrespective of agonist type, the Bz-sensitive NaCl CT response demonstrated a maximum response enhancement limit of about 75% over control value.     

The Staphylococcus aureus NuoL-Like Protein MpsA Contributes to the Generation of Membrane Potential

In aerobic microorganisms, the entry point of respiratory electron transfer is represented by the NADH:quinone oxidoreductase. The enzyme couples the oxidation of NADH with the reduction of quinone. In the type 1 NADH:quinone oxidoreductase (Ndh1), this reaction is accompanied by the translocation of cations, such as H⁺ or Na⁺. In Escherichia coli, cation translocation is accomplished by the subunit NuoL, thus generating membrane potential (Δψ). Some microorganisms achieve NADH oxidation by the alternative, nonelectrogenic type 2 NADH:quinone oxidoreductase (Ndh2), which is not cation translocating. Since these enzymes had not been described in Staphylococcus aureus, the goal of this study was to identify proteins operating in the NADH:quinone segment of its respiratory chain. We demonstrated that Ndh2 represents a NADH:quinone oxidoreductase in S. aureus. Additionally, we identified a hypothetical protein in S. aureus showing sequence similarity to the proton-translocating subunit NuoL of complex I in E. coli: the NuoL-like protein MpsA. Mutants with deletion of the nuoL-like gene mpsA and its corresponding operon, mpsABC (mps for membrane potential-generating system), exhibited a small-colony-variant-like phenotype and were severely affected in Δψ and oxygen consumption rates. The MpsABC proteins did not confer NADH oxidation activity. Using an Na⁺/H⁺ antiporter-deficient E. coli strain, we could show that MpsABC constitute a cation-translocating system capable of Na⁺ transport. Our study demonstrates that MpsABC represent an important functional system of the respiratory chain of S. aureus that acts as an electrogenic unit responsible for the generation of Δψ.     

Diurnal changes in volume and solute transport coefficients of phaseolus roots

Volume (J_v) and solute (J_s) fluxes through Phaseolus root systems were observed over a 24-hour period. The volume flux was varied in a pressure chamber by altering the hydrostatic pressure in 10 steps, from 0 to 0.41 megapascals. All root systems showed strong diurnal peaks in volume flux. The five transport coefficients (σ, ω, J_s^*, L_p, and π^*) were estimated from a nonlinear least squares algorithm. Analysis of the data revealed that all the coefficients exhibited a diurnal rhythm. When the total differential of the volume flux was considered it was possible to show that the diurnal changes in volume flux were due to a complex interaction between the diurnally shifting coefficients with the role of each highly dependent on the level of volume flux. At low volume fluxes, ω, J_s^*, and π^* accounted for nearly all the diurnal change in volume flux. At high volume fluxes, however, the major influence shifted to L_p and π^*, while ω and J_s^* became relatively unimportant. Thus, π^* was the only coefficient of interest across the entire range of J_v and appeared to be the single most important one in determining the diurnal rhythm of J_v under conditions of a constant applied pressure.     

Vasotocin has the potential to inhibit basolateral Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-pump current across isolated skin of tree frog in vitro<Emphasis Type="Italic">,</Emphasis> via its V<Subscript>2</Subscript>-type receptor/cAMP pathway

Adult frog skin transports Na⁺ from the apical to the basolateral side across the skin. Antidiuretic hormone (ADH) is involved in the regulation of Na⁺ transport in both mammals and amphibians. We investigated the effect of arginine vasotocin (AVT), the ADH of amphibians, on the short-circuit current (SCC) across intact skin and on the basolateral Na⁺/K⁺-pump current across apically nystatin-permeabilized skin of the tree frog, Hyla japonica, in which the V₂-type ADH receptor is expressed in vitro. In intact skin, 1 pM AVT had no effect on the SCC, but 10 nM AVT was sufficient to stimulate the SCC since 10 nM and 1 μM of AVT increased the SCC 3.2- and 3.4-fold, respectively (P > 0.9). However, in permeabilized skin, AVT (1 μM) decreased the Na⁺/K⁺-pump current to 0.79 times vehicle control. Similarly, 500 μM of 8Br-cAMP increased the SCC 3.2-fold, yet 1 mM of 8Br-cAMP decreased the Na⁺/K⁺-pump current to 0.76 times vehicle control. Arachidonic acid (10⁻⁵ M) tended to decrease the Na⁺/K⁺-pump current. To judge from these in vitro experiments, AVT has the potential to inhibit the basolateral Na⁺/K⁺-pump current via the V₂-type receptor/cAMP pathway in the skin of the tree frog.     

Gating of Single N-type Calcium Channels Recorded from Bullfrog Sympathetic Neurons

For many neurons, N-type calcium channels provide the primary pathway for calcium influx during an action potential. We investigated the gating properties of single N-type calcium channels using the cell-attached patch technique. With 100 mM Ba²⁺ in the pipet, mean N-channel open probability (P _o, measured over 100 ms) increased with depolarization, but the range at a single voltage was large (e.g., P _o at +40 mV ranged from 0.1 to 0.8). The open dwell time histograms were generally well fit by a single exponential with mean open time (τ_o) increasing from 0.7 ms at +10 mV to 3.1 ms at +40 mV. Shut time histograms were well fit by two exponentials. The brief shut time component (τ_sh1 = 0.3 ms) did not vary with the test potential, while the longer shut time component (τ_sh2) decreased with voltage from 18.9 ms at +10 mV to 2.3 ms at +40 mV. Although N-channel P _o during individual sweeps at +40 mV was often high (∼0.8), mean P _o was reduced by null sweeps, low P _o gating, inactivation, and slow activation. The variability in mean P _o across patches resulted from differences in the frequency these different gating processes were expressed by the channels. Runs analysis showed that null sweeps tended to be clustered in most patches, but that inactivating and slowly activating sweeps were generally distributed randomly. Low P _o gating (P _o = 0.2, τ_o = 1 ms at +40 mV) could be sustained for ∼1 min in some patches. The clustering of null sweeps and sweeps with low P _o gating is consistent with the idea that they result from different modes of N-channel gating. While P _o of the main N-channel gating state is high, the net P _o is reduced to a maximum value of close to 0.5 by other gating processes.     

Melting studies of dangling-ended DNA hairpins: effects of end length,loop sequence and biotinylation of loop bases

The effects of 3′ single-strand dangling-ends of different lengths, sequence identity of hairpin loop, and hairpin loop biotinylation at different loop residues on DNA hairpin thermodynamic stability were investigated. Hairpins contained 16 bp stem regions and five base loops formed from the sequence, 5′-TAGTCGACGTGGTCC-N₅-GGACCACGTCGACTAG-E_n-3′. The length of the 3′ dangling-ends (E_n) was n = 13 or 22 bases. The identities of loop bases at positions 2 and 4 were varied. Biotinylation was varied at loop base positions 2, 3 or 4. Melting buffers contained 25 or 115 mM Na⁺. Average t_m values for all molecules were 73.5 and 84.0°C in 25 and 115 mM Na⁺, respectively. Average two-state parameters evaluated from van’t Hoff analysis of the melting curve shapes in 25 mM Na⁺ were ΔH_vH = 84.8 ± 15.5 kcal/mol, ΔS_vH = 244.8 ± 45.0 cal/K·mol and ΔG_vH = 11.9 ± 2.1 kcal/mol. In 115 mM Na⁺, two-state parameters were not very different at ΔH_vH = 80.42 ± 12.74 kcal/mol, ΔS_vH = 225.24 ± 35.88 cal/K·mol and ΔG_vH = 13.3 ± 2.0 kcal/mol. Differential scanning calorimetry (DSC) was performed to test the validity of the two-state assumption and evaluated van’t Hoff parameters. Thermodynamic parameters from DSC measurements (within experimental error) agreed with van’t Hoff parameters, consistent with a two-state process. Overall, dangling-end DNA hairpin stabilities are not affected by dangling-end length, loop biotinylation or sequence and vary uniformly with [Na⁺]. Consider able freedom is afforded when designing DNA hairpins as probes in nucleic acid based detection assays, such as microarrays.