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Ernst Bamberg - One of the best experts on this subject based on the ideXlab platform.

  • sensory rhodopsin ii from the haloalkaliphilic natronobacterium pharaonis light activated proton transfer reactions
    Biophysical Journal, 2000
    Co-Authors: Georg Schmies, Beate Luttenberg, Igor Chizhov, Martin Engelhard, A Becker, Ernst Bamberg
    Abstract:

    In the present work the light-activated proton transfer reactions of sensory rhodopsin II from Natronobacterium pharaonis (pSRII) and those of the channel-mutants D75N-pSRII and F86D-pSRII are investigated using flash photolysis and Black Lipid Membrane (BLM) techniques. Whereas the photocycle of the F86D-pSRII mutant is quite similar to that of the wild-type protein, the photocycle of D75N-pSRII consists of only two intermediates. The addition of external proton donors such as azide, or in the case of F86D-pSRII, imidazole, accelerates the reprotonation of the Schiff base, but not the turnover. The electrical measurements prove that pSRII and F86D-pSRII can function as outwardly directed proton pumps, whereas the mutation in the extracellular channel (D75N-pSRII) leads to an inwardly directed transient current. The almost negligible size of the photostationary current is explained by the long-lasting photocycle of about a second. Although the M decay, but not the photocycle turnover, of pSRII and F86D-pSRII is accelerated by the addition of azide, the photostationary current is considerably increased. It is discussed that in a two-photon process a late intermediate (N- and/or O-like species) is photoconverted back to the original resting state; thereby the long photocycle is cut short, giving rise to the large increase of the photostationary current. The results presented in this work indicate that the function to generate ion gradients across Membranes is a general property of archaeal rhodopsins.

  • different modes of proton translocation by sensory rhodopsin i
    The EMBO Journal, 1996
    Co-Authors: Ulrich Haupts, Ernst Bamberg, Dieter Oesterhelt
    Abstract:

    Abstract The Membrane-bound complex between sensory rhodopsin I (SRI) and its transducer HtrI forms the functional photoreceptor unit that allows transmission of light signals to the flagellar motor. Although being a photosensor, SRI, the mutant SRI-D76N and the HtrI-SRI complex can transport protons, as we demonstrate by using the sensitive and ion-specific Black Lipid Membrane technique. SRI sustains an orange light-driven (one-photon-driven) outward proton transport which is enhanced by additional blue light (two-photon-driven). The vectoriality of the two-photon-driven transport could be reversed at neutral pH from the outward to the inward direction by switching the cut-off wavelength of the long wavelength light from 550 to 630 nm. The cut-off wavelength determining the reversal point decreases with decreasing pH. The currents could be enhanced by azide. A two-photon-driven inward proton transport by SRI-D76N (catalyzed by azide) and by the complex HtrI-SRI is demonstrated. The influence of pH and azide concentration on the rise and decay kinetics of the SRI380 intermediate is analyzed. The different modes of proton translocation of the SRI species are discussed on the basis of a general model of proton translocation of retinal proteins and in the context of signal transduction.

  • charge transport by ion translocating Membrane proteins on solid supported Membranes
    Biophysical Journal, 1993
    Co-Authors: Karsten Seifert, Klaus Fendler, Ernst Bamberg
    Abstract:

    A new method for the investigation of ion translocating Membrane proteins is presented. Protein containing Membrane fragments or vesicles are adsorbed to a solid supported Membrane. The solid supported Membrane consists of a Lipid monolayer on a gold evaporated or gold sputtered glass substrate which is coated with a long chained mercaptan (CH3(CH2)mSH, m = 15, 17). Specific conductance and specific capacitance of the solid supported Membrane are comparable to those of a Black Lipid Membrane. However, the solid supported Membrane has the advantage of a much higher mechanical stability. The electrical activity of bacteriorhodopsin, Na,K-ATPase, H,K-ATPase, and Ca-ATPase on the solid supported Membrane is measured and compared to signals obtained on a conventionally prepared Black Lipid Membrane. It is shown that both methods yield similar results. The solid supported Membrane therefore represents an alternative method for the investigation of electrical properties of ion translocating transMembrane proteins.

Yoshiro Yonezawa - One of the best experts on this subject based on the ideXlab platform.

  • photochemical regulation of ion transport through quasi channels embedded in Black Lipid Membrane
    Materials Science and Engineering: C, 1997
    Co-Authors: Motomu Tanaka, Yoshiro Yonezawa
    Abstract:

    Abstract The Black Lipid Membranes (BLM) of soybean lecithin doped with an amphiphilic azobenzene derivative, 4-octyl-4′-(5-carboxypentamethyleneoxy) azobenzene (8A5), was fabricated, and the K+ ion permeation through the “quasi-channels”, as represented by the steady d.c. current signal across the Membrane, was photochemically regulated. A transient photocurrent overlapping with the steady d.c. current due to photoisomerization of 8A5 was observed in the BLM cell filled with a diluted KCl solution for the first time. Dependence of the steady photocurrent on the molar concentration ratio of 8 A5 to lecithin, [8 A5] / [lecithin], was also examined. It has been found that the normalized conductivity change, δG/G(trans), is roughly proportional to the square of [8A5]/[lecithin]. The result would suggest that one “quasichannel” consists of two 8A5 molecules.

  • Photochemical Switching of Ion Transport Through ‘Quasi-Channels’ Incorporated into Black Lipid Membrane
    Molecular Crystals and Liquid Crystals Science and Technology. Section A. Molecular Crystals and Liquid Crystals, 1997
    Co-Authors: Motomu Tanaka, Yoshiro Yonezawa
    Abstract:

    Abstract We fabricated the Black Lipid Membrane (BLM) of soybean lecithin doped with spiropyran derivatives. When a positive DC voltage was applied on the BLM, the Membrane current changed under alternate irradiation with ultraviolet light and visible light. The change in the Membrane conductivity due to photoisomerization is responsible to the change in the ion permeability across the Membrane. The photoresponsive BLM cell exhibited nonsymmetric current-voltage characteristics, which suggest the localization of spiropyran molecules on one side of the Membrane.

Patrick Augustijns - One of the best experts on this subject based on the ideXlab platform.

  • Application of PAMPA-models to predict BBB permeability including efflux ratio, plasma protein binding and physicochemical parameters
    International journal of pharmaceutics, 2010
    Co-Authors: J. Mensch, Libuse Jaroskova L, Wendy E. Sanderson, A. Melis, Claire Mackie, Geert Verreck, Marcus E. Brewster, Patrick Augustijns
    Abstract:

    Abstract This study examines whether algorithms to predict brain penetration of 88 drug candidates could benefit from inclusion of PAMPA data such as P eff , flux and Membrane retention. Specifically the ability to fit experimentally derived LogBB data with PAMPA information and compound related physicochemical and structural parameters was assessed. Collected data were analyzed by partial least square analysis and various regression models for LogBB. Four PAMPA methodologies were evaluated in this study including: (1) a PAMPA-BLM (Black Lipid Membrane) model, (2) a PAMPA-DS (double sink) model, (3) a PAMPA-BBB (blood–brain barrier) model and (4) a PAMPA-BBB-UWL (unstirred water layer). Additionally, plasma protein binding (PPB) experiments and a Caco-2 assay were performed to determine the unbound fraction in plasma and the efflux ratio, respectively, for subsets of the selected compounds. This information was combined with the obtained PAMPA data in an effort to improve the predictions of LogBB. Taken in aggregate, the results presented, suggest that the PAMPA-BLM parameters are the most important contributors to predict the LogBB. The optimized multiple linear regression (MLR) relationship including the PAMPA-BLM properties demonstrated a slightly improved prediction compared to the model without the PAMPA-BLM parameters. Including the plasma protein binding of 15 compounds resulted in a significantly improved PAMPA-BLM prediction of LogBB, while integrating the efflux ratio with PAMPA-BLM or PAMPA-BBB P eff values, resulted in improved classification of brain permeable [BBB + (LogBB ≥ 0)] and impermeable [BBB − (LogBB

  • evaluation of various pampa models to identify the most discriminating method for the prediction of bbb permeability
    European Journal of Pharmaceutics and Biopharmaceutics, 2010
    Co-Authors: J. Mensch, A. Melis, Claire Mackie, Geert Verreck, Marcus E. Brewster, Patrick Augustijns
    Abstract:

    The Parallel Artificial Membrane Permeability Assay (PAMPA) has been successfully introduced into the pharmaceutical industry to allow useful predictions of passive oral absorption. Over the last 5 years, researchers have modified the PAMPA such that it can also evaluate passive blood-brain barrier (BBB) permeability. This paper compares the permeability of 19 structurally diverse, commercially available drugs assessed in four different PAMPA models: (1) a PAMPA-BLM (Black Lipid Membrane) model, (2) a PAMPA-DS (Double Sink) model, (3) a PAMPA-BBB model and (4) a PAMPA-BBB-UWL (unstirred water layer) model in order to find the most discriminating method for the prediction of BBB permeability. Both the PAMPA-BBB model and the PAMPA-BLM model accurately identified compounds which pass the BBB (BBB+) and those which poorly penetrate the BBB (BBB-). For these models, BBB+ and BBB- classification ranges, in terms of permeability values, could be defined, offering the opportunity to validate the paradigm with in vivo data. The PAMPA models were subsequently applied to a set of 14 structurally diverse internal JJ BBB-: LogBB<0). PAMPA-BLM resulted in three false positive identifications, while PAMPA-BBB misclassified only one compound. Additionally, a Caco-2 assay was performed to determine the efflux ratio of all compounds in the test set. The false positive that occurred in both models was shown to be related to an increased efflux ratio. Both the PAMPA-BLM and the PAMPA-BBB models can be used to predict BBB permeability of compounds in combination with an assay that provides p-gp efflux data, such as the Caco-2 assay.

Motomu Tanaka - One of the best experts on this subject based on the ideXlab platform.

  • photochemical regulation of ion transport through quasi channels embedded in Black Lipid Membrane
    Materials Science and Engineering: C, 1997
    Co-Authors: Motomu Tanaka, Yoshiro Yonezawa
    Abstract:

    Abstract The Black Lipid Membranes (BLM) of soybean lecithin doped with an amphiphilic azobenzene derivative, 4-octyl-4′-(5-carboxypentamethyleneoxy) azobenzene (8A5), was fabricated, and the K+ ion permeation through the “quasi-channels”, as represented by the steady d.c. current signal across the Membrane, was photochemically regulated. A transient photocurrent overlapping with the steady d.c. current due to photoisomerization of 8A5 was observed in the BLM cell filled with a diluted KCl solution for the first time. Dependence of the steady photocurrent on the molar concentration ratio of 8 A5 to lecithin, [8 A5] / [lecithin], was also examined. It has been found that the normalized conductivity change, δG/G(trans), is roughly proportional to the square of [8A5]/[lecithin]. The result would suggest that one “quasichannel” consists of two 8A5 molecules.

  • Photochemical Switching of Ion Transport Through ‘Quasi-Channels’ Incorporated into Black Lipid Membrane
    Molecular Crystals and Liquid Crystals Science and Technology. Section A. Molecular Crystals and Liquid Crystals, 1997
    Co-Authors: Motomu Tanaka, Yoshiro Yonezawa
    Abstract:

    Abstract We fabricated the Black Lipid Membrane (BLM) of soybean lecithin doped with spiropyran derivatives. When a positive DC voltage was applied on the BLM, the Membrane current changed under alternate irradiation with ultraviolet light and visible light. The change in the Membrane conductivity due to photoisomerization is responsible to the change in the ion permeability across the Membrane. The photoresponsive BLM cell exhibited nonsymmetric current-voltage characteristics, which suggest the localization of spiropyran molecules on one side of the Membrane.

Dieter Oesterhelt - One of the best experts on this subject based on the ideXlab platform.

  • different modes of proton translocation by sensory rhodopsin i
    The EMBO Journal, 1996
    Co-Authors: Ulrich Haupts, Ernst Bamberg, Dieter Oesterhelt
    Abstract:

    Abstract The Membrane-bound complex between sensory rhodopsin I (SRI) and its transducer HtrI forms the functional photoreceptor unit that allows transmission of light signals to the flagellar motor. Although being a photosensor, SRI, the mutant SRI-D76N and the HtrI-SRI complex can transport protons, as we demonstrate by using the sensitive and ion-specific Black Lipid Membrane technique. SRI sustains an orange light-driven (one-photon-driven) outward proton transport which is enhanced by additional blue light (two-photon-driven). The vectoriality of the two-photon-driven transport could be reversed at neutral pH from the outward to the inward direction by switching the cut-off wavelength of the long wavelength light from 550 to 630 nm. The cut-off wavelength determining the reversal point decreases with decreasing pH. The currents could be enhanced by azide. A two-photon-driven inward proton transport by SRI-D76N (catalyzed by azide) and by the complex HtrI-SRI is demonstrated. The influence of pH and azide concentration on the rise and decay kinetics of the SRI380 intermediate is analyzed. The different modes of proton translocation of the SRI species are discussed on the basis of a general model of proton translocation of retinal proteins and in the context of signal transduction.