Hv1 Proton Channel Opening Is Preceded by a Voltage-independent Transition

The voltage sensing domain (VSD) of the voltage-gated proton channel Hv1 mediates a H⁺-selective conductance that is coordinately controlled by the membrane potential (V) and the transmembrane pH gradient (ΔpH). Allosteric control of Hv1 channel opening by ΔpH (V-ΔpH coupling) is manifested by a characteristic shift of approximately 40 mV per ΔpH unit in the activation. To further understand the mechanism for V-ΔpH coupling in Hv1, H⁺ current kinetics of activation and deactivation in excised membrane patches were analyzed as a function of the membrane potential and the pH in the intracellular side of the membrane (pH_I). In this study, it is shown for the first time to our knowledge that the opening of Hv1 is preceded by a voltage-independent transition. A similar process has been proposed to constitute the step involving coupling between the voltage-sensing and pore domains in tetrameric voltage-gated channels. However, for Hv1, the VSD functions as both the voltage sensor and the conduction pathway, suggesting that the voltage independent transition is intrinsic to the voltage-sensing domain. Therefore, this article proposes that the underlying mechanism for the activation of Hv1 involves a process similar to VSD relaxation, a process previously described for voltage-gated channels and voltage-controlled enzymes. Finally, deactivation seemingly occurs as a strictly voltage dependent process, implying that the kinetic event leading to opening of the proton conductance are different than those involved in the closing. Thus, from this work it is proposed that Hv1 activity displays hysteresis.     

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.     

Analysis of ionic channels by a flash spectrophotometric technique applicable to thylakoid membranes: CF0, the proton channel of the chloroplast ATP synthase,and, for comparison,gramicidin

Summary We previously introduced a flash spectrophotometric method to analyze proton conduction by CF₀ in vesicles derived from thylakoid membranes (H. Lill, S. Engelbrecht, G. Schönknecht & W. Junge, 1986,Eur. J. Biochem. 160:627–634). The unit conductance of CF₀, as revealed by this technique, was orders of magnitude higher than that theoretically expected for a hydrogen-bonded chain. We scrutinized the validity of this method. Small vesicles were derived from thylakoids by EDTA treatment. The intrinsic electric generators in the membrane were stimulated by short flashes of light and the relaxation of the voltage via ionic channels was measured through electrochromic absorption changes of intrinsic pigments. The voltage decay was stimulated by a statistical model. As the vesicle-size distribution had only a minor influence, the simulation required only two fit parameters, the first proportional to the unit conductance of an active channelG, and the second denoting the average number of active channels per vesiclen. This technique was applied to CF₀, the proton channel of the chloroplast ATP synthase, and to gramicidin, serving as a standard. For both channels we found the above two fit parameters physically meaningful. They could be independently varied in predictable wasy, i.e.n by addition of known inhibitors of F₀-type proton channels andG via the temperature. for gramicidin, the unit conductance (2.7 pS) was within the range described in the literature. This established the competence of this method for studies on the mechanism of proton conduction by CF₀, whose conductance so far has not been accessible to other, more conventional approaches. The time-averaged unit conductance of CF₀ was about 1 pS, equivalent to the turnover of 6×10⁵ H⁺/(CF₀·sec) at 100 mV driving force.     

Proton channel of the chloroplast ATP synthase,CF0: Its time-averaged single-channel conductance as function of pH,temperature, isotopic and ionic medium composition

Summary The proton-driven ATP synthase of chloroplasts is composed of two elements, CF₀ and CF₁. The membrane bound CF₀ conducts protons and the peripheral CF₁ interacts with nucleotides. By flash spectrophotometric techniques applied to thylakoid membranes from which about 50% of total CF₁ was removed, we have previously determined the protonic (timeaveraged) single-channel conductance of CF₀. Being in the order of 1 pS, it was sufficiently large to support the proposed role of CF₀ as a low-impedance access for protons to the coupling site in CF₀CF₁. On the other hand, it was too large to be readily reconciled with current concepts of proton supply to and proton conduction through the channel.We studied the time-averaged single-channel conductance of CF₀ under variation of pH, pD, ionic composition, temperature, and water/membrane structure with the following results: (i) CF₀ was proton-specific even against a background of 300mm monovalent or 30mm divalent catins. (ii) While the conductance of CF₀ was pH/pD-independent in the range from 5.6–8.0, in D₂O it was lower by a constant factor of 1.7 than in H₂O (iii) Addition of glycerol diminished the conductance and abolished the isotope effect. (iv) The Arrhenius activation energy was 42 kJ/mol and thus intermediate between the ones found for the water-filled pore, gramicidin (30 kJ/mol), and the mobile carrier, valinomycin (65 kJ/mol).The results implied that CF₀ is endowed with an extremely proton-specific (10⁷-fold) selectivity filter. Its conductance is very high, and its conduction cycle is not necessarily rate limited by a protolytic reaction. The mechanisms of rapid proton supply to the channel mouth and of proton conduction remained enigmatic.     

Hv1 Proton Channel Opening Is Preceded by a Voltage-independent Transition

The voltage sensing domain (VSD) of the voltage-gated proton channel Hv1 mediates a H⁺-selective conductance that is coordinately controlled by the membrane potential (V) and the transmembrane pH gradient (ΔpH). Allosteric control of Hv1 channel opening by ΔpH (V-ΔpH coupling) is manifested by a characteristic shift of approximately 40 mV per ΔpH unit in the activation. To further understand the mechanism for V-ΔpH coupling in Hv1, H⁺ current kinetics of activation and deactivation in excised membrane patches were analyzed as a function of the membrane potential and the pH in the intracellular side of the membrane (pH_I). In this study, it is shown for the first time to our knowledge that the opening of Hv1 is preceded by a voltage-independent transition. A similar process has been proposed to constitute the step involving coupling between the voltage-sensing and pore domains in tetrameric voltage-gated channels. However, for Hv1, the VSD functions as both the voltage sensor and the conduction pathway, suggesting that the voltage independent transition is intrinsic to the voltage-sensing domain. Therefore, this article proposes that the underlying mechanism for the activation of Hv1 involves a process similar to VSD relaxation, a process previously described for voltage-gated channels and voltage-controlled enzymes. Finally, deactivation seemingly occurs as a strictly voltage dependent process, implying that the kinetic event leading to opening of the proton conductance are different than those involved in the closing. Thus, from this work it is proposed that Hv1 activity displays hysteresis.     

Temperature Dependence of Voltage-gated H+ Currents in Human Neutrophils,Rat Alveolar Epithelial Cells,and Mammalian Phagocytes

H⁺ currents in human neutrophils, rat alveolar epithelial cells, and several mammalian phagocyte cell lines were studied using whole-cell and excised-patch tight-seal voltage clamp techniques at temperatures between 6 and 42°C. Effects of temperature on gating kinetics were distinguished from effects on the H⁺ current amplitude. The activation and deactivation of H⁺ currents were both highly temperature sensitive, with a Q ₁₀ of 6–9 (activation energy, E _a, ≈ 30–38 kcal/mol), greater than for most other ion channels. The similarity of E _a for channel opening and closing suggests that the same step may be rate determining. In addition, when the turn-on of H⁺ currents with depolarization was fitted by a delay and single exponential, both the delay and the time constant (τ_act) had similarly high Q ₁₀. These results could be explained if H⁺ channels were composed of several subunits, each of which undergoes a single rate-determining gating transition. H⁺ current gating in all mammalian cells studied had similarly strong temperature dependences. The H⁺ conductance increased markedly with temperature, with Q ₁₀ ≥ 2 in whole-cell experiments. In excised patches where depletion would affect the measurement less, the Q ₁₀ was 2.8 at >20°C and 5.3 at <20°C. This temperature sensitivity is much greater than for most other ion channels and for H⁺ conduction in aqueous solution, but is in the range reported for H⁺ transport mechanisms other than channels; e.g., carriers and pumps. Evidently, under the conditions employed, the rate-determining step in H⁺ permeation occurs not in the diffusional approach but during permeation through the channel itself. The large E _a of permeation intrinsically limits the conductance of this channel, and appears inconsistent with the channel being a water-filled pore. At physiological temperature, H⁺ channels provide mammalian cells with an enormous capacity for proton extrusion.     

Single-Channel Kinetics,Inactivation, and Spatial Distribution of Inositol Trisphosphate (IP3) Receptors in Xenopus Oocyte Nucleus

Single-channel properties of the Xenopus inositol trisphosphate receptor (IP₃R) ion channel were examined by patch clamp electrophysiology of the outer nuclear membrane of isolated oocyte nuclei. With 140 mM K⁺ as the charge carrier (cytoplasmic [IP₃] = 10 μM, free [Ca²⁺] = 200 nM), the IP₃R exhibited four and possibly five conductance states. The conductance of the most-frequently observed state M was 113 pS around 0 mV and ∼300 pS at 60 mV. The channel was frequently observed with high open probability (mean P _o = 0.4 at 20 mV). Dwell time distribution analysis revealed at least two kinetic states of M with time constants τ < 5 ms and ∼20 ms; and at least three closed states with τ ∼1 ms, ∼10 ms, and >1 s. Higher cytoplasmic potential increased the relative frequency and τ of the longest closed state. A novel “flicker” kinetic mode was observed, in which the channel alternated rapidly between two new conductance states: F₁ and F₂. The relative occupation probability of the flicker states exhibited voltage dependence described by a Boltzmann distribution corresponding to 1.33 electron charges moving across the entire electric field during F₁ to F₂ transitions. Channel run-down or inactivation (τ ∼ 30 s) was consistently observed in the continuous presence of IP₃ and the absence of change in [Ca²⁺]. Some (∼10%) channel disappearances could be reversed by an increase in voltage before irreversible inactivation. A model for voltage-dependent channel gating is proposed in which one mechanism controls channel opening in both the normal and flicker modes, whereas a separate independent mechanism generates flicker activity and voltage- reversible inactivation. Mapping of functional channels indicates that the IP₃R tends to aggregate into microscopic (<1 μm) as well as macroscopic (∼10 μm) clusters. Ca²⁺-independent inactivation of IP₃R and channel clustering may contribute to complex [Ca²⁺] signals in cells.     

Survival of Probiotic Lactobacilli in Acidic Environments Is Enhanced in the Presence of Metabolizable Sugars

Lactobacillus rhamnosus GG is an industrially significant probiotic strain with proven health benefits. In this study, the effect of glucose on L. rhamnosus GG survival was analyzed in simulated gastric juice at pH 2.0. It was found that the presence of 19.4 mM glucose resulted in up to 6-log₁₀-enhanced survival following 90 min of exposure. Further work with dilute HCl confirmed that glucose was the sole component responsible. Comparative analysis with other Lactobacillus strains revealed that enhanced survival was apparent in all strains, but at different pH values. The presence of glucose at concentrations from 1 to 19.4 mM enhanced L. rhamnosus GG survival from 6.4 to 8 log₁₀ CFU ml⁻¹ in simulated gastric juice. The mechanisms behind the protective effect of glucose were investigated. Addition of N′,N′-dicyclohexylcarbodiimide to simulated gastric juice caused survival to collapse, which was indicative of a prominent role in inhibition of F₀F₁-ATPase. Further work with neomycin-resistant mutants that exhibited 38% to 48% of the F₀F₁-ATPase activity of the parent confirmed this, as the survival in the presence of glucose of these mutants decreased 3 × 10⁶-fold compared with the survival of the wild type (which had a viability of 8.02 log₁₀ CFU ml⁻¹). L. rhamnosus GG survival in acidic conditions occurred only in the presence of sugars that it could metabolize efficiently. To confirm the involvement of glycolysis in the glucose effect, iodoacetic acid was used to inhibit glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity. The reduction in GAPDH activity caused survival to decrease by 8.30 log₁₀ CFU ml⁻¹ in the presence of glucose. The data indicate that glucose provides ATP to F₀F₁-ATPase via glycolysis, enabling proton exclusion and thereby enhancing survival during gastric transit.     

A Highly Temperature-sensitive Proton Current in Mouse Bone Marrow–derived Mast Cells

Proton (H⁺) conductive pathways are suggested to play roles in the regulation of intracellular pH. We characterized temperature-sensitive whole cell currents in mouse bone marrow–derived mast cells (BMMC), immature proliferating mast cells generated by in vitro culture. Heating from 24 to 36°C reversibly and repeatedly activated a voltage-dependent outward conductance with Q₁₀ of 9.9 ± 3.1 (mean ± SD) (n = 6). Either a decrease in intracellular pH or an increase in extracellular pH enhanced the amplitude and shifted the activation voltage to more negative potentials. With acidic intracellular solutions (pH 5.5), the outward current was detected in some cells at 24°C and Q₁₀ was 6.0 ± 2.6 (n = 9). The reversal potential was unaffected by changes in concentrations of major ionic constituents (K⁺, Cl⁻, and Na⁺), but depended on the pH gradient, suggesting that H⁺ (equivalents) is a major ion species carrying the current. The H⁺ current was featured by slow activation kinetics upon membrane depolarization, and the activation time course was accelerated by increases in depolarization, elevating temperature and extracellular alkalization. The current was recorded even when ATP was removed from the intracellular solution, but the mean amplitude was smaller than that in the presence of ATP. The H⁺ current was reversibly inhibited by Zn²⁺ but not by bafilomycin A₁, an inhibitor for a vacuolar type H⁺-ATPase. Macroscopic measurements of pH using a fluorescent dye (BCECF) revealed that a rapid recovery of intracellular pH from acid-load was attenuated by lowering temperature, addition of Zn²⁺, and depletion of extracellular K⁺, but not by bafilomycin A₁. These results suggest that the H⁺ conductive pathway contributes to intracellular pH homeostasis of BMMC and that the high activation energy may be involved in enhancement of the H⁺ conductance.     

State Transitions in the Green Alga Scenedesmus Obliquus Probed by Time-Resolved Chlorophyll Fluorescence Spectroscopy and Global Data Analysis

Decay-associated fluorescence spectra of the green alga Scenedesmus obliquus have been measured by single-photon timing with picosecond resolution in various states of light adaptation. The data have been analyzed by applying a global data analysis procedure. The amplitudes of the decay-associated spectra allow a determination of the relative antenna sizes of the photosystems. We arrive at the following conclusions: (a) The fluorescence kinetics of algal cells with open PS II centers (F₀ level) have to be described by a sum of three exponential components. These decay components are attributed to photosystem (PS) I (τ ≈ 85 ps, λ_max^em ≈ 695-700 nm), open PS II α-centers (τ ≈ 300 ps, λ_max^em = 685 nm), and open PS II β-centers (τ ≈ 600 ps, λ_max^em = 685 nm). A fourth component of very low amplitude (τ ≈ 2.2-2.3 ns, λ_max^em = 685 nm) derives from dead chlorophyll. (b) At the F_max level of fluorescence there are also three decay components. They originate from PS I with properties identical to those at the F₀ level, from closed PS II α-centers (τ ≈ 2.2 ns, λ_max^em = 685 nm) and from closed PS β-centers (τ ≈ 1.2 ns, λ_max^em = 685 nm). (c) The major effect of light-induced state transitions on the fluorescence kinetics involves a change in the relative antenna size of α- and β-units brought about by the reversible migration of light-harvesting complexes between α-centers and β-centers. (d) A transition to state II does not measurably increase the direct absorption cross-section (antenna size) of PS I. Our data can be rationalized in terms of a model of the antenna organization that relates the effects of state transitions and light-harvesting complex phosphorylation with the concepts of PS II α,β-heterogeneity. We discuss why our results are in disagreement with those of a recent lifetime study of Chlorella by M. Hodges and I. Moya (1986, Biochim. Biophys. Acta., 849:193-202).     

Electrodiffusion,Barrier, and Gating Analysis of DIDS-insensitive Chloride Conductance in Human Red Blood Cells Treated with Valinomycin or Gramicidin

Current-voltage curves for DIDS-insensitive Cl⁻ conductance have been determined in human red blood cells from five donors. Currents were estimated from the rate of cell shrinkage using flow cytometry and differential laser light scattering. Membrane potentials were estimated from the extracellular pH of unbuffered suspensions using the proton ionophore FCCP. The width of the Gaussian distribution of cell volumes remained invariant during cell shrinkage, indicating a homogeneous Cl⁻ conductance among the cells. After pretreatment for 30 min with DIDS, net effluxes of K⁺ and Cl⁻ were induced by valinomycin and were measured in the continued presence of DIDS; inhibition was maximal at ∼65% above 1 μM DIDS at both 25°C and 37°C. The nonlinear current-voltage curves for DIDS-insensitive net Cl⁻ effluxes, induced by valinomycin or gramicidin at varied [K⁺]_o, were compared with predictions based on (1) the theory of electrodiffusion, (2) a single barrier model, (3) single occupancy, multiple barrier models, and (4) a voltage-gated mechanism. Electrodiffusion precisely describes the relationship between the measured transmembrane voltage and [K⁺]_o. Under our experimental conditions (pH 7.5, 23°C, 1–3 μM valinomycin or 60 ng/ml gramicidin, 1.2% hematocrit), the constant field permeability ratio P_K/P_Cl is 74 ± 9 with 10 μM DIDS, corresponding to 73% inhibition of P_Cl. Fitting the constant field current-voltage equation to the measured Cl⁻ currents yields P _Cl = 0.13 h⁻¹ with DIDS, compared to 0.49 h⁻¹ without DIDS, in good agreement with most previous studies. The inward rectifying DIDS-insensitive Cl⁻ current, however, is inconsistent with electrodiffusion and with certain single-occupancy multiple barrier models. The data are well described either by a single barrier located near the center of the transmembrane electric field, or, alternatively, by a voltage-gated channel mechanism according to which the maximal conductance is 0.055 ± 0.005 S/g Hb, half the channels are open at −27 ± 2 mV, and the equivalent gating charge is −1.2 ± 0.3.     

Basic Properties of Rotary Dynamics of the Molecular Motor Enterococcus hirae V1-ATPase

V-ATPases are rotary molecular motors that generally function as proton pumps. We recently solved the crystal structures of the V₁ moiety of Enterococcus hirae V-ATPase (EhV₁) and proposed a model for its rotation mechanism. Here, we characterized the rotary dynamics of EhV₁ using single-molecule analysis employing a load-free probe. EhV₁ rotated in a counterclockwise direction, exhibiting two distinct rotational states, namely clear and unclear, suggesting unstable interactions between the rotor and stator. The clear state was analyzed in detail to obtain kinetic parameters. The rotation rates obeyed Michaelis-Menten kinetics with a maximal rotation rate (V_max) of 107 revolutions/s and a Michaelis constant (K_m) of 154 μm at 26 °C. At all ATP concentrations tested, EhV₁ showed only three pauses separated by 120°/turn, and no substeps were resolved, as was the case with Thermus thermophilus V₁-ATPase (TtV₁). At 10 μm ATP (⪡K_m), the distribution of the durations of the ATP-waiting pause fit well with a single-exponential decay function. The second-order binding rate constant for ATP was 2.3 × 10⁶ m⁻¹ s⁻¹. At 40 mm ATP (⪢K_m), the distribution of the durations of the catalytic pause was reproduced by a consecutive reaction with two time constants of 2.6 and 0.5 ms. These kinetic parameters were similar to those of TtV₁. Our results identify the common properties of rotary catalysis of V₁-ATPases that are distinct from those of F₁-ATPases and will further our understanding of the general mechanisms of rotary molecular motors.     

Simultaneous Measurements of Cytoplasmic K Concentration and the Plasma Membrane Electrical Parameters in Single Membrane Samples of Chara corallina

The electrophysiological properties of cytoplasm-rich fragments (single membrane samples) prepared from internodal cells of Chara corallina were explored in conjunction with K⁺-sensitive microelectrode and current-voltage (I-V) measurements. This system eliminated the problem of the inaccessible cytoplasmic layer, while preserving many of the electrical characteristics of the intact cells. In 0.1 millimolar external K concentration (K_o⁺), the resting conductance (membrane conductance G_m, 0.85 ± 0.25 Siemens per square meter (±standard error)) of the single membrane samples, was dominated by the proton pump, as suggested by the response of the near-linear I-V characteristic to changes in external pH. Initial cytoplasmic K⁺ activities (a_K+), judged most reliable, gave values of 117 ± 67 millimolar; stable a_K+ values were 77 ± 31 millimolar. Equilibrium potentials for K⁺ (Nernst equilibrium potential) (E_K) calculated, using either of these data sets, were near the mean membrane potential (V_m). On a cell-to-cell basis, however, E_K was generally negative of the V_m, despite an electrogenic contribution from the Chara proton pump. When K_o⁺ was increased to 1.0 millimolar or above, G_m rose (by 8- to 10-fold in 10 millimolar K_o⁺), the steady state I-V characteristics showed a region of negative slope conductance, and V_m followed E_K. These results confirm previous studies which implicated a K_o⁺-induced and voltage-dependent permeability to K⁺ at the Chara plasma membrane. They provide an explanation for transitions between apparent K_o⁺-insensitive and K_o⁺-sensitive (`K⁺ electrode') behavior displayed by the membrane potential, as recorded in many algae and higher plant cells.     

Halide Permeation in Wild-Type and Mutant Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels

Permeation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channels by halide ions was studied in stably transfected Chinese hamster ovary cells by using the patch clamp technique. In cell-attached patches with a high Cl⁻ pipette solution, the CFTR channel displayed outwardly rectifying currents and had a conductance near the membrane potential of 6.0 pS at 22°C or 8.7 pS at 37°C. The current–voltage relationship became linear when patches were excised into symmetrical, N-tris(hydroxymethyl)methyl-2-aminomethane sulfonate (TES)-buffered solutions. Under these conditions, conductance increased from 7.0 pS at 22°C to 10.9 pS at 37°C. The conductance at 22°C was ∼1.0 pS higher when TES and HEPES were omitted from the solution, suggesting weak, voltage-independent block by pH buffers. The relationship between conductance and Cl⁻ activity was hyperbolic and well fitted by a Michaelis-Menten–type function having a K _m of ∼38 mM and maximum conductance of 10 pS at 22°C. Dilution potentials measured with NaCl gradients indicated high anion selectivity (P_Na/P_Cl = 0.003–0.028). Biionic reversal potentials measured immediately after exposure of the cytoplasmic side to various test anions indicated P_I (1.8) > P_Br (1.3) > P_Cl (1.0) > P_F (0.17), consistent with a “weak field strength” selectivity site. The same sequence was obtained for external halides, although inward F⁻ flow was not observed. Iodide currents were protocol dependent and became blocked after 1–2 min. This coincided with a large shift in the (extrapolated) reversal potential to values indicating a greatly reduced I⁻/Cl⁻ permeability ratio (P_I/P_Cl < 0.4). The switch to low I⁻ permeability was enhanced at potentials that favored Cl⁻ entry into the pore and was not observed in the R347D mutant, which is thought to lack an anion binding site involved in multi-ion pore behavior. Interactions between Cl⁻ and I⁻ ions may influence I⁻ permeation and be responsible for the wide range of P_I/P_Cl ratios that have been reported for the CFTR channel. The low P_I/P_Cl ratio usually reported for CFTR only occurred after entry into an altered permeability state and thus may not be comparable with permeability ratios for other anions, which are obtained in the absence of iodide. We propose that CFTR displays a “weak field strength” anion selectivity sequence.     

Single-Molecule Analysis of the Rotation of F1-ATPase under High Hydrostatic Pressure

F₁-ATPase is the water-soluble part of ATP synthase and is an ATP-driven rotary molecular motor that rotates the rotary shaft against the surrounding stator ring, hydrolyzing ATP. Although the mechanochemical coupling mechanism of F₁-ATPase has been well studied, the molecular details of individual reaction steps remain unclear. In this study, we conducted a single-molecule rotation assay of F₁ from thermophilic bacteria under various pressures from 0.1 to 140 MPa. Even at 140 MPa, F₁ actively rotated with regular 120° steps in a counterclockwise direction, showing high conformational stability and retention of native properties. Rotational torque was also not affected. However, high hydrostatic pressure induced a distinct intervening pause at the ATP-binding angles during continuous rotation. The pause was observed under both ATP-limiting and ATP-saturating conditions, suggesting that F₁ has two pressure-sensitive reactions, one of which is evidently ATP binding. The rotation assay using a mutant F₁(βE190D) suggested that the other pressure-sensitive reaction occurs at the same angle at which ATP binding occurs. The activation volumes were determined from the pressure dependence of the rate constants to be +100 Å³ and +88 Å³ for ATP binding and the other pressure-sensitive reaction, respectively. These results are discussed in relation to recent single-molecule studies of F₁ and pressure-induced protein unfolding.     

Membrane-Bound ATPase Contributes to Hop Resistance of Lactobacillus brevis

The activity of the membrane-bound H⁺-ATPase of the beer spoilage bacterium Lactobacillus brevis ABBC45 increased upon adaptation to bacteriostatic hop compounds. The ATPase activity was optimal around pH 5.6 and increased up to fourfold when L. brevis was exposed to 666 μM hop compounds. The extent of activation depended on the concentration of hop compounds and was maximal at the highest concentration tested. The ATPase activity was strongly inhibited by N,N′-dicyclohexylcarbodiimide, a known inhibitor of F_oF₁-ATPase. Western blots of membrane proteins of L. brevis with antisera raised against the α- and β-subunits of F_oF₁-ATPase from Enterococcus hirae showed that there was increased expression of the ATPase after hop adaptation. The expression levels, as well as the ATPase activity, decreased to the initial nonadapted levels when the hop-adapted cells were cultured further without hop compounds. These observations strongly indicate that proton pumping by the membrane-bound ATPase contributes considerably to the resistance of L. brevis to hop compounds.     

Subunit δ Is the Key Player for Assembly of the H+-translocating Unit of Escherichia coli FOF1 ATP Synthase

The ATP synthase (F_OF₁) of Escherichia coli couples the translocation of protons across the cytoplasmic membrane to the synthesis or hydrolysis of ATP. This nanomotor is composed of the rotor c₁₀γϵ and the stator ab₂α₃β₃δ. To study the assembly of this multimeric enzyme complex consisting of membrane-integral as well as peripheral hydrophilic subunits, we combined nearest neighbor analyses by intermolecular disulfide bond formation or purification of partially assembled F_OF₁ complexes by affinity chromatography with the use of mutants synthesizing different sets of F_OF₁ subunits. Together with a time-delayed in vivo assembly system, the results demonstrate that F_OF₁ is assembled in a modular way via subcomplexes, thereby preventing the formation of a functional H⁺-translocating unit as intermediate product. Surprisingly, during the biogenesis of F_OF₁, F₁ subunit δ is the key player in generating stable F_O. Subunit δ serves as clamp between ab₂ and c₁₀α₃β₃γϵ and guarantees that the open H⁺ channel is concomitantly assembled within coupled F_OF₁ to maintain the low membrane proton permeability essential for viability, a general prerequisite for the assembly of multimeric H⁺-translocating enzymes.     

pH-Induced Proton Permeability Changes of Plasma Membrane Vesicles

In vivo studies with leaf cells of aquatic plant species such as Elodea nuttallii revealed the proton permeability and conductance of the plasma membrane to be strongly pH dependent. The question was posed if similar pH dependent permeability changes also occur in isolated plasma membrane vesicles. Here we report the use of acridine orange to quantify passive proton fluxes. Right-side out vesicles were exposed to pH jumps. From the decay of the applied ΔpH the proton fluxes and proton permeability coefficients (P_H+) were calculated. As in the intact Elodea plasma membrane, the proton permeability of the vesicle membrane is pH sensitive, an effect of internal pH as well as external pH on P_H+ was observed. Under near symmetric conditions, i.e., zero electrical potential and zero ΔpH, P_H+ increased from 65 × 10⁻⁸ at pH 8.5 to 10⁻¹ m/sec at pH 11 and the conductance from 13 × 10⁻⁶ to 30 × 10⁻⁴ S/m². At a constant pH _i of 8 and a pH _o going from 8.5 to 11, P_H+ increased more than tenfold from 2 to 26 × 10⁻⁶ m/sec. The calculated values of P_H+ were several orders of magnitude lower than those obtained from studies on intact leaves. Apparently, in plasma membrane purified vesicles the transport system responsible for the observed high proton permeability in vivo is either (partly) inactive or lost during the procedure of vesicle preparation. The residue proton permeability is in agreement with values found for liposome or planar lipid bilayer membranes, suggesting that it reflects an intrinsic permeability of the phospholipid bilayer to protons. Possible implications of these findings for transport studies on similar vesicle systems are discussed. Received: 5 April 1995/Revised: 28 March 1996     

Proton conductance caused by long-chain fatty acids in phospholipid bilayer membranes

Summary Mechanisms of proton conductance (G _H) were investigated in phospholipid bilayer membranes containing long-chain fatty acids (lauric, myristic, palmitic, oleic or phytanic). Membranes were formed from diphytanoyl phosphatidylcholine in decane plus chlorodecane (usually 30% vol/vol). Fatty acids were added either to the aqueous phase or to the membrane-forming solution. Proton conductance was calculated from the steadystate total conductance and the H⁺ diffusion potential produced by a transmembrane pH gradient. Fatty acids causedG _H to increase in proportion to the first power of the fatty acid concentration. TheG _H induced by fatty acids was inhibited by phloretin, low pH and serum albumin.G _H was increased by chlorodecane, and the voltage dependence ofG _H was superlinear. The results suggest that fatty acids act as simple (A^– type) proton carriers. The membrane: water partition coefficient (K _p ) and adsorption coefficient () were estimated by finding the membrane and aqueous fatty acid concentrations which gave identical values ofG _H. For palmitic and oleic acidsK _p was about 10⁵ and was about 10^–2 cm. The A^– translocation or flip-flop rate (k _a ) was estimated from the value ofG _H and the fatty acid concentration in the membrane, assuming that A^– translocation was the rate limiting step in H⁺ transport. Thek _A 's were about 10^–4 sec^–1, slower than classical weak-acid uncouplers by a factor of 10⁵. Although long-chain fatty acids are relatively inefficient H⁺ carriers, they may cause significant biological H⁺ conductance when present in the membrane at high concentrations, e.g., in ischemia, hypoxia, hormonally induced lipolysis, or certain hereditary disorders, e.g., Refsum's (phytanic acid storage) disease.     

Δψ and ΔpH are equivalent driving forces for proton transport through isolated F0 complexes of ATP synthases

The membrane-embedded F₀ part of ATP synthases is responsible for ion translocation during ATP synthesis and hydrolysis. Here, we describe an in vitro system for measuring proton fluxes through F₀ complexes by fluorescence changes of the entrapped fluorophore pyranine. Starting from purified enzyme, the F₀ part was incorporated unidirectionally into phospholipid vesicles. This allowed analysis of proton transport in either synthesis or hydrolysis direction with Δψ or ΔpH as driving forces. The system displayed a high signal-to-noise ratio and can be accurately quantified. In contrast to ATP synthesis in the Escherichia coli F₁F₀ holoenzyme, no significant difference was observed in the efficiency of ΔpH or Δψ as driving forces for H⁺-transport through F₀. Transport rates showed linear dependency on the driving force. Proton transport in hydrolysis direction was about 2400 H⁺/(s × F₀) at Δψ of 120 mV, which is approximately twice as fast as in synthesis direction. The chloroplast enzyme was faster and catalyzed H⁺-transport at initial rates of 6300 H⁺/(s × F₀) under similar conditions. The new method is an ideal tool for detailed kinetic investigations of the ion transport mechanism of ATP synthases from various organisms.