Local Control Model of Excitation–Contraction Coupling in Skeletal Muscle

The voltage-activated H⁺ selective conductance of rat alveolar epithelial cells was studied using whole-cell and excised-patch voltage-clamp techniques. The effects of substituting deuterium oxide, D₂O, for water, H₂O, on both the conductance and the pH dependence of gating were explored. D⁺ was able to permeate proton channels, but with a conductance only about 50% that of H⁺. The conductance in D₂O was reduced more than could be accounted for by bulk solvent isotope effects (i.e., the lower mobility of D⁺ than H⁺), suggesting that D⁺ interacts specifically with the channel during permeation. Evidently the H⁺ or D⁺ current is not diffusion limited, and the H⁺ channel does not behave like a water-filled pore. This result indirectly strengthens the hypothesis that H⁺ (or D⁺) and not OH⁻ is the ionic species carrying current. The voltage dependence of H⁺ channel gating characteristically is sensitive to pH_o and pH_i and was regulated by pD_o and pD_i in an analogous manner, shifting 40 mV/U change in the pD gradient. The time constant of H⁺ current activation was about three times slower (τ_act was larger) in D₂O than in H₂O. The size of the isotope effect is consistent with deuterium isotope effects for proton abstraction reactions, suggesting that H⁺ channel activation requires deprotonation of the channel. In contrast, deactivation (τ_tail) was slowed only by a factor ≤1.5 in D₂O. The results are interpreted within the context of a model for the regulation of H⁺ channel gating by mutually exclusive protonation at internal and external sites (Cherny, V.V., V.S. Markin, and T.E. DeCoursey. 1995. J. Gen. Physiol. 105:861–896). Most of the kinetic effects of D₂O can be explained if the pK _a of the external regulatory site is ∼0.5 pH U higher in D₂O.     

On the Divalent Metal Ion Dependence of DNA Cleavage by Restriction Endonucleases of the EcoRI Family

Restriction endonucleases of the PD…D/EXK family need Mg²⁺ for DNA cleavage. Whereas Mg²⁺ (or Mn²⁺) promotes catalysis, Ca²⁺ (without Mg²⁺) only supports DNA binding. The role of Mg²⁺ in DNA cleavage by restriction endonucleases has elicited many hypotheses, differing mainly in the number of Mg²⁺ involved in catalysis. To address this problem, we measured the Mg²⁺ and Mn²⁺ concentration dependence of DNA cleavage by BamHI, BglII, Cfr10I, EcoRI, EcoRII (catalytic domain), MboI, NgoMIV, PspGI, and SsoII, which were reported in co-crystal structure analyses to bind one (BglII and EcoRI) or two (BamHI and NgoMIV) Me²⁺ per active site. DNA cleavage experiments were carried out at various Mg²⁺ and Mn²⁺ concentrations at constant ionic strength. All enzymes show a qualitatively similar Mg²⁺ and Mn²⁺ concentration dependence. In general, the Mg²⁺ concentration optimum (between ∼ 1 and 10 mM) is higher than the Mn²⁺ concentration optimum (between ∼ 0.1 and 1 mM). At still higher Mg²⁺ or Mn²⁺ concentrations, the activities of all enzymes tested are reduced but can be reactivated by Ca²⁺. Based on these results, we propose that one Mg²⁺ or Mn²⁺ is critical for restriction enzyme activation, and binding of a second Me²⁺ plays a role in modulating the activity. Steady-state kinetics carried out with EcoRI and BamHI suggest that binding of a second Mg²⁺ or Mn²⁺ mainly leads to an increase in K_m, such that the inhibitory effect of excess Mg²⁺ or Mn²⁺ can be overcome by increasing the substrate concentration. Our conclusions are supported by molecular dynamics simulations and are consistent with the structural observations of both one and two Me²⁺ binding to these enzymes.     

Free energy profiles for H conduction in the D-pathway of Cytochrome c Oxidase: A study of the wild type and N98D mutant enzymes

The molecular mechanism for proton conduction in the D-pathway of Cytochrome c Oxidase (CcO) is investigated through the free energy profile, i.e., potential of mean force (PMF) calculations of both the native enzyme and the N98D mutant. The multistate empirical valence bond (MS-EVB) model was applied to simulate the interaction of an excess proton with the channel environment. In the study of the wild type enzyme, the PMF reveals the previously proposed proton trap inside the channel; it also shows a high free energy barrier against the passage of proton at the entry of the channel, where two conserved asparagines (ASN80/98) may be essential for the gating of proton uptake. We also present data from an investigation of the N98D mutant, which has been previously shown to completely eliminate proton pumping but significantly enhance the oxidase activity in Rhodobacter sphaeroides. These results suggest that mutating Asn98 to negatively charged aspartate will create an unfavorable energy barrier sufficiently high to prevent the overall proton uptake through the D-pathway, whereas with a protonated aspartic acid the proton conduction was found to be accelerated. Plausible explanations for the origin of the uncoupling of proton pumping from the oxidase activity will be discussed.     

Simulating redox coupled proton transfer in cytochrome c oxidase: looking for the proton bottleneck

Gaining a detailed understanding of the molecular nature of the redox coupled proton transfer in cytochrome c oxidase (COX) is one of the challenges of modern biophysics. The present work addresses this by integrating approaches for simulations of proton transport (PTR) and electron transfer (ET). The resulting method converts the electrostatic energies of different charge configurations and reorganization energies to free-energy profiles for different PTR and ET pathways. This approach provides for the first time a tool to study the actual activation barriers and kinetics of different feasible PTR processes in the cycle of COX. Using this tool, we explore the PTR through the bottleneck water molecules. It is found that a stepwise PTR along this commonly assumed path leads to far too high barriers and is, thus, inconsistent with the observed kinetics. Furthermore, the simulated free-energy profile does not provide a simple gating mechanism. Fortunately, we obtain reasonable kinetics when we consider a PTR that involves a concerted transfer of protons to and from E286. Finally, semi-qualitative considerations of the forward and backward barriers point toward open questions about the actual gating process and offer a feasible pumping mechanism. Although further studies are clearly needed, we believe that our approach offers a general and effective tool for correlating the structure of COX with its function.     

Concerted action of two cation filters in the aquaporin water channel

Aquaporin (AQP) facilitated water transport is common to virtually all cell membranes and is marked by almost perfect specificity and high flux rates. Simultaneously, protons and cations are strictly excluded to maintain ionic transmembrane gradients. Yet, the AQP cation filters have not been identified experimentally. We report that three point mutations turned the water-specific AQP1 into a proton/alkali cation channel with reduced water permeability and the permeability sequence: H⁺ ≫K⁺ >Rb⁺ >Na⁺ >Cs⁺ >Li⁺. Contrary to theoretical models, we found that electrostatic repulsion at the central asn-pro-ala (NPA) region does not suffice to exclude protons. Full proton exclusion is reached only in conjunction with the aromatic/arginine (ar/R) constriction at the pore mouth. In contrast, alkali cations are blocked by the NPA region but leak through the ar/R constriction. Expression of alkali-leaking AQPs depolarized membrane potentials and compromised cell survival. Our results hint at the alkali-tight but solute-unselective NPA region as a feature of primordial channels and the proton-tight and solute-selective ar/R constriction variants as later adaptations within the AQP superfamily.     

The involvement of aquaglyceroporins in transport of boron in barley roots

Boron (B) enters cells as the uncharged boric acid, a small neutral molecule with sufficient lipid solubility to cross cell membranes without the aid of transport proteins. The extent to which the observed uptake rates for B in plants can be explained by this simple physical process was examined by applying treatments expected to inhibit the membrane transporters most likely to be involved in B transport. These experiments established that at least 50% of B uptake could be facilitated by transporters. The B transport characteristics of two barley aquaglyceroporins, HvPIP1;3 and HvPIP1;4, were investigated using yeast complementation assays. Expression of both genes in yeast resulted in increased B sensitivity. Transport assays in yeast confirmed that HvPIP1;3 and HvPIP1;4 are both capable of transporting B. The physiological role of these HvPIP1 genes in B transport is uncertain since their expression was not responsive to B nutritional status, and they continued to be expressed under toxicity conditions.     

How fast monoamine oxidases decompose adrenaline? Kinetics of isoenzymes A and B evaluated by empirical valence bond simulation

This work scrutinizes kinetics of decomposition of adrenaline catalyzed by monoamine oxidase (MAO) A and B enzymes, a process controlling the levels of adrenaline in the central nervous system and other tissues. Experimental kinetic data for MAO A and B catalyzed decomposition of adrenaline are reported only in the form of the maximum reaction rate. Therefore, we estimated the experimental free energy barriers form the kinetic data of closely related systems using regression method, as was done in our previous study. By using multiscale simulation on the Empirical Valence Bond (EVB) level, we studied the chemical reactivity of the MAO A catalyzed decomposition of adrenaline and we obtained a value of activation free energy of 17.3 ± 0.4 kcal/mol. The corresponding value for MAO B is 15.7 ± 0.7 kcal/mol. Both values are in good agreement with the estimated experimental barriers of 16.6 and 16.0 kcal/mol for MAO A and MAO B, respectively. The fact that we reproduced the kinetic data and preferential catalytic effect of MAO B over MAO A gives additional support to the validity of the proposed hydride transfer mechanism. Furthermore, we demonstrate that adrenaline is preferably involved in the reaction in a neutral rather than in a protonated form due to considerably higher barriers computed for the protonated adrenaline substrate. The results are discussed in the context of chemical mechanism of MAO enzymes and possible applications of multiscale simulation to rationalize the effects of MAO activity on adrenaline level.     

Enhancement of proton conductance by mutations of the selectivity filter of aquaporin-1

Prevention of cation permeation in wild-type aquaporin-1 (AQP1) is believed to be associated with the Asn-Pro-Ala (NPA) region and the aromatic/arginine selectivity filter (SF) domain. Previous work has suggested that the NPA region helps to impede proton permeation due to the protein backbone collective macrodipoles that create an environment favoring a directionally discontinuous channel hydrogen-bonded water chain and a large electrostatic barrier. The SF domain contributes to the proton permeation barrier by a spatial restriction mechanism and direct electrostatic interactions. To further explore these various effects, the free-energy barriers and the maximum cation conductance for the permeation of various cations through the AQP1-R195V and AQP1-R195S mutants are predicted computationally. The cations studied included the hydrated excess proton that utilizes the Grotthuss shuttling mechanism, a model “classical” charge localized hydronium cation that exhibits no Grotthuss shuttling, and a sodium cation. The hydrated excess proton was simulated using a specialized multi-state molecular dynamics method including a proper physical treatment of the proton shuttling and charge defect delocalization. Both AQP1 mutants exhibit a surprising cooperative effect leading to a reduction in the free-energy barrier for proton permeation around the NPA region due to altered water configurations in the SF region, with AQP1-R195S having a higher conductance than AQP1-R195V. The theoretical predictions are experimentally confirmed in wild-type AQP1 and the mutants expressed in Xenopus oocytes. The combined results suggest that the SF domain is a specialized structure that has evolved to impede proton permeation in aquaporins.     

Kir4.1 channels regulate swelling of astroglial processes in experimental spinal cord edema

In glial cells, inwardly rectifying K⁺ channels (Kir) control extracellular [K⁺]_o homeostasis by uptake of K⁺ from the extracellular space and release of K⁺ into the microvasculature. Kir channels were also recently implicated in K⁺-associated water influx and cell swelling. We studied the time-dependent expression and functional implication of the glial Kir4.1 channel for astroglial swelling in a spinal cord edema model. In this CNS region, Kir4.1 is expressed on astrocytes from the second postnatal week on and co-localizes with aquaporin 4 (AQP4). Swelling of individual astrocytes in response to osmotic stress and to pharmacological Kir blockade were analyzed by time-lapse-two-photon laser-scanning microscopy in situ . Application of 30% hypotonic solution induced astroglial soma swelling whereas no swelling was observed on astroglial processes or endfeet. Co-application of hypotonic solution and Ba²⁺, a Kir channel blocker, induced prominent swelling of astroglial processes. In Kir4.1−/− mice, however, somatic as well as process swelling was observed upon application of 30% hypotonic solutions. No additional effect was provoked upon co-application with Ba²⁺. Our experiments show that Kir channels prevent glial process swelling under osmotic stress. The underlying Kir channel subunit that controls glial process swelling is Kir4.1, whereas changes of the glial soma are not substantially related to Kir4.1.     

Proton transfer along the external proton channel of bacteriorhodopsin

The process of proton transfer along a proton channel is considered using bacteriorhodopsin as a model system, for which a large body of experimental data is available. The possible amino acid composition of the external proton half-channel of bacteriorhodopsin and the stepwise scheme of proton transfer consistent with experimental data are proposed. The rate of proton transfer between fixed centers is assessed for certain regions of this channel for which spectroscopic data are available.     

植物水孔蛋白的亚细胞分布与生理功能研究浅析

水孔蛋白(aquaporin,AQP)因具有水转运活性而得名,然而随着研究的深入,水孔蛋白转运活性的多样性与生理功能的多样性不断被报道．本文综合分析了植物水孔蛋白亚细胞定位与功能多样性的研究进展,重点综述了植物水孔蛋白广泛的亚细胞分布特点,以及亚细胞上的再分布现象与植物水孔蛋白生理功能多样性间的关系,并对植物水孔蛋白研究中存在的 问题及研究方向进行了分析,认为水孔蛋白多样化的生理功能的作用机制需要结合其组织定位与亚细胞定位进行分析才能 揭示．     

Plasma Membrane Water Permeability of Cultured Cells and Epithelia Measured by Light Microscopy with Spatial Filtering

A method was developed to measure the osmotic water permeability (P_f) of plasma membranes in cell layers and applied to cells and epithelia expressing molecular water channels. It was found that the integrated intensity of monochromatic light in a phase contrast or dark field microscope was dependent on relative cell volume. For cells of different size and shape (Sf9, MDCK, CHO, A549, tracheal epithelia, BHK), increased cell volume was associated with decreased signal intensity; generally the signal decreased 10–20% for a twofold increase in cell volume. A theory relating signal intensity to relative cell volume was developed based on spatial filtering and changes in optical path length associated with cell volume changes. Theory predictions were confirmed by signal measurements of cell layers bathed in solutions of various osmolarities and refractive indices. The excellent signal-to-noise ratio of the transmitted light detection permitted measurement of cell volume changes of <1%. The method was applied to characterize transfected cells and tissues that natively express water channels. P_f in control Chinese hamster ovary cells was low (0.0012 cm/s at 23°C) and increased more than fourfold upon stable transfection with aquaporins 1, 2, 4, or 5. P_f in apical and basolateral membranes in polarized epithelial cells grown on porous supports was measured. P_f ^bl and P_f ^ap were 0.0011 and 0.0024 cm/s (MDCK cells), and 0.0039 and 0.0052 cm/s (human tracheal cells) at 23°C. In intact toad urinary bladder, basolateral P_f was 0.036 cm/s and apical membrane P_f after vasopressin stimulation was 0.025 cm/s at 23°C. The results establish light microscopy with spatial filtering as a technically simple and quantitative method to measure water permeability in cell layers and provide the first measurement of the apical and basolateral membrane permeabilities of several important epithelial cell types.     

Differential Induction of Two Glucoamylases in Candida pelliculosa

Rapid growth of the submerged shoots of deepwater rice is essential for survival during the rainy season. We investigated changes in the expression of vacuolar H⁺-ATPase (V-ATPase), H⁺-pyrophosphatase (V-PPase), and aquaporins under submerged conditions. The amounts of vacuolar proton pumps, which support the active transport of ions into the vacuoles, were maintained on a membrane protein basis in the developing vacuoles. Among the six isogenes of V-PPase, OsVHP1;3 was markedly enhanced by submersion. The gene expression of efficient water channels, OsTIP1;1, OsTIP2;2, OsPIP1;1, OsPIP2;1, and OsPIP2;2, was markedly enhanced by submersion. The increase in aquaporin expression might support quick elongation of internodes. The mRNA levels of OsNIP2;2 and OsNIP3;1, which transport silicic and boric acids respectively, clearly decreased. The present study indicates that internodes of deepwater rice upregulate vacuolar proton pumps and water channel aquaporins and downregulate aquaporins that allow permeation of the substrates that suppress internode growth.