The Experts below are selected from a list of 267 Experts worldwide ranked by ideXlab platform

Klaus Fendler - One of the best experts on this subject based on the ideXlab platform.

  • transport mechanism and ph regulation of the na h antiporter nhaa from escherichia coli an Electrophysiological study
    Journal of Biological Chemistry, 2011
    Co-Authors: Thomas Mager, Abraham Rimon, Etana Padan, Klaus Fendler
    Abstract:

    Using an Electrophysiological Assay the activity of NhaA was tested in a wide pH range from pH 5.0 to 9.5. Forward and reverse transport directions were investigated at zero membrane potential using preparations with inside-out and right side-out-oriented transporters with Na+ or H+ gradients as the driving force. Under symmetrical pH conditions with a Na+ gradient for activation, both the wt and the pH-shifted G338S variant exhibit highly symmetrical transport activity with bell-shaped pH dependences, but the optimal pH was shifted 1.8 pH units to the acidic range in the variant. In both strains the pH dependence was associated with a systematic increase of the Km for Na+ at acidic pH. Under symmetrical Na+ concentration with a pH gradient for NhaA activation, an unexpected novel characteristic of the antiporter was revealed; rather than being down-regulated, it remained active even at pH as low as 5. These data allowed a transport mechanism to advance based on competing Na+ and H+ binding to a common transport site and a kinetic model to develop quantitatively explaining the experimental results. In support of these results, both alkaline pH and Na+ induced the conformational change of NhaA associated with NhaA cation translocation as demonstrated here by trypsin digestion. Furthermore, Na+ translocation was found to be associated with the displacement of a negative charge. In conclusion, the Electrophysiological Assay allows the revelation of the mechanism of NhaA antiport and sheds new light on the concept of NhaA pH regulation.

  • Robust Electrophysiological Assays using Solid Supported Membranes: the Organic Cation Transporter OCT2
    Australian Journal of Chemistry, 2011
    Co-Authors: Olga Gaiko, Ingo G. Janausch, Sven Geibel, Henning Vollert, Petra Arndt, Sigrid Gonski, Klaus Fendler
    Abstract:

    An Electrophysiological Assay platform based on solid supported membranes (SSM) for the organic cation transporter (OCT) is presented. Stable Chinese hamster ovary (CHO) cell lines overexpressing the human (hOCT2) and rat transporters (rOCT2) were generated and validated. Membrane preparations from the cell lines were investigated using SSM-based electrophysiology. Baculovirus transfected insect cells (HighFive and Mimic Sf9) were also tested with the same Assay but yielded less than optimal results. The Assays were validated by the determination of substrate affinities and inhibition by standard inhibitors. The study demonstrates the suitability of the SSM-based Electrophysiological OCT Assay for rapid and automatic screening of drug candidates.

  • the g215r mutation in the cl h antiporter clc 7 found in ado ii osteopetrosis does not abolish function but causes a severe trafficking defect
    PLOS ONE, 2010
    Co-Authors: Patrick Schulz, Johannes Werner, Tobias Stauber, Kim Henriksen, Klaus Fendler
    Abstract:

    Background ClC-7 is a ubiquitous transporter which is broadly expressed in mammalian tissues. It is implied in the pathogenesis of lysosomal storage disease and osteopetrosis. Because of its endosomal/lysosomal localization it is still poorly characterized. Methodology/Principal Findings An Electrophysiological characterization of rat ClC-7 using solid-supported membrane-based electrophysiology is presented. The measured currents show the characteristics of ClC-7 and confirm its function as a Cl−/H+-antiporter. We have used rat ClC-7 in CHO cells as a model system to investigate the functionality and cellular localization of the wt transporter and its variant G213R ClC-7 which is the analogue of human G215R ClC-7 responsible for autosomal dominant osteopetrosis type II. Our study shows that rat G213R ClC-7 is functional but has a localization defect in CHO cells which prevents it from being correctly targeted to the lysosomal membrane. The Electrophysiological Assay is tested as a tool for drug discovery. The Assay is validated with a number of drug candidates. It is shown that ClC-7 is inhibited by DIDS, NPPB and NS5818 at micromolar concentrations. Conclusions/Significance It is suggested that the scenario found in the CHO model system also applies to the human transporter and that mislocalization rather than impaired functionality of G215R ClC-7 is the primary cause of the related autosomal dominant osteopetrosis type II. Furthermore, the robust solid-supported membrane-based Electrophysiological Assay is proposed for rapid screening for potential ClC-7 inhibitors which are discussed for treatment of osteoporosis.

  • An automatic Electrophysiological Assay for the neuronal glutamate transporter mEAAC1.
    Journal of neuroscience methods, 2008
    Co-Authors: Robin Krause, Natalie Watzke, Bela Kelety, Wolfgang Dörner, Klaus Fendler
    Abstract:

    A rapid and robust Electrophysiological Assay based on solid supported membranes (SSM) for the murine neuronal glutamate transporter mEAAC1 is presented. Measurements at different concentrations revealed the EAAC1 specific affinities for l-glutamate (K(m)=24microM), l-aspartate (K(m)=5microM) and Na(+) (K(m)=33mM) and an inhibition constant K(i) for dl-threo-beta-benzyloxyaspartic acid (TBOA) of 1microM. Inhibition by 3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-6-carboxylic acid (HIP-B) was not purely competitive with an IC(50) of 13microM. Experiments using SCN(-) concentration jumps yielded large transient currents in the presence of l-glutamate showing the characteristics of the glutamate-gated anion conductance of EAAC1. Thus, SSM-based electrophysiology allows the analysis of all relevant transport modes of the glutamate transporter on the same sample. K(+) and Na(+) gradients could be applied to the transporter. Experiments in the presence and absence of Na(+) and K(+) gradients demonstrated that the protein is still able to produce a charge translocation when no internal K(+) is present. In this case, the signal amplitude is smaller and a lower apparent affinity for l-glutamate of 144microM is found. Finally the Assay was adapted to a commercial fully automatic system for SSM-based electrophysiology and was validated by determining the substrate affinities and inhibition constants as for the laboratory setup. The combination of automatic function and its ability to monitor all transport modes of EAAC1 make this system an universal tool for industrial drug discovery.

Roderick Mackinnon - One of the best experts on this subject based on the ideXlab platform.

  • Cryo-EM Structure of a KCNQ1/CaM Complex Reveals Insights into Congenital Long QT Syndrome.
    Cell, 2017
    Co-Authors: Ji Sun, Roderick Mackinnon
    Abstract:

    KCNQ1 is the pore-forming subunit of cardiac slow-delayed rectifier potassium (IKs) channels. Mutations in the kcnq1 gene are the leading cause of congenital long QT syndrome (LQTS). Here, we present the cryoelectron microscopy (cryo-EM) structure of a KCNQ1/calmodulin (CaM) complex. The conformation corresponds to an "uncoupled," PIP2-free state of KCNQ1, with activated voltage sensors and a closed pore. Unique structural features within the S4-S5 linker permit uncoupling of the voltage sensor from the pore in the absence of PIP2. CaM contacts the KCNQ1 voltage sensor through a specific interface involving a residue on CaM that is mutated in a form of inherited LQTS. Using an Electrophysiological Assay, we find that this mutation on CaM shifts the KCNQ1 voltage-activation curve. This study describes one physiological form of KCNQ1, depolarized voltage sensors with a closed pore in the absence of PIP2, and reveals a regulatory interaction between CaM and KCNQ1 that may explain CaM-mediated LQTS.

  • Structure of the cytoplasmic beta subunit-T1 assembly of voltage-dependent K+ channels.
    Science (New York N.Y.), 2000
    Co-Authors: Jacqueline M. Gulbis, Ming Zhou, Sabine Mann, Roderick Mackinnon
    Abstract:

    The structure of the cytoplasmic assembly of voltage-dependent K+ channels was solved by x-ray crystallography at 2.1 angstrom resolution. The assembly includes the cytoplasmic (T1) domain of the integral membrane alpha subunit together with the oxidoreductase beta subunit in a fourfold symmetric T1(4)beta4 complex. An Electrophysiological Assay showed that this complex is oriented with four T1 domains facing the transmembrane pore and four beta subunits facing the cytoplasm. The transmembrane pore communicates with the cytoplasm through lateral, negatively charged openings above the T1(4)beta4 complex. The inactivation peptides of voltage-dependent K(+) channels reach their site of action by entering these openings.

  • Purification, characterization, and synthesis of an inward-rectifier K+ channel inhibitor from scorpion venom.
    Biochemistry, 1997
    Co-Authors: Roderick Mackinnon
    Abstract:

    We have purified a protein inhibitor of an inward-rectifier K+ channel, ROMK1, from the venom of the scorpion Leiurus quinquestriatus var. hebraeus. The inhibitor is Lq2, a previously discovered blocker of voltage- and Ca2+-activated K+ channels. Mutations were made on the channel and the inhibitor, and the resulting effects were examined using an Electrophysiological Assay. The data show that Lq2 blocks the pore of ROMK1, and that the interaction surface on Lq2 is the same for binding to inward-rectifier, voltage-activated, or Ca2+-activated K+ channels. These findings support the notion that different classes of K+ channels have different gates but a similar K+-selective pore structure.

Mark R. Bowlby - One of the best experts on this subject based on the ideXlab platform.

  • validation of a medium throughput Electrophysiological Assay for kcnq2 3 channel enhancers using ionworks ht
    Journal of Biomolecular Screening, 2007
    Co-Authors: Flora Jow, Ru Shen, Pranab K. Chanda, Eugene Tseng, Howard Zhang, Jeffrey D. Kennedy, John Dunlop, Mark R. Bowlby
    Abstract:

    Enhancers of KCNQ channels are known to be effective in chronic pain models. To discover novel enhancers of KCNQ channels, the authors developed a medium-throughput Electrophysiological Assay by using the IonWorks platform. Screening of 20 CHOK1 clones stably expressing KCNQ2/3 was performed on the IonWorks HT until the best clone (judged from seal rate, current level, and stability) was obtained. The KCNQ2/3 current amplitude in the cells was found to increase from 60 ± 15 pA to 473 ± 80 pA (at –10 mV), and the expression rate was increased by 56% when the cells were incubated at 27 °C overnight. The clone used for compound screening had a seal rate of greater than 90% and an overall success rate of greater than 70%. The voltage step protocol (hold cells at –80 mV and depolarize to –10 mV for 1 s) was designed to provide moderate current but still allow for pharmacological current enhancement. EC 50 s were generated from 8-point concentration-response curves with a control compound on each plate using compounds that were also tested with conventional patch clamp. The authors found that there was a very good correlation (R 2 > 0.9) between the 2 Assays, thus demonstrating the highly predictive nature of the IonWorks Assay. (Journal of Biomolecular Screening 2007;1059-1067)

  • Validation of a Medium-Throughput Electrophysiological Assay for KCNQ2/3 Channel Enhancers Using IonWorks HT
    Journal of biomolecular screening, 2007
    Co-Authors: Flora Jow, Ru Shen, Pranab K. Chanda, Eugene Tseng, Howard Zhang, Jeffrey D. Kennedy, John Dunlop, Mark R. Bowlby
    Abstract:

    Enhancers of KCNQ channels are known to be effective in chronic pain models. To discover novel enhancers of KCNQ channels, the authors developed a medium-throughput Electrophysiological Assay by using the IonWorks platform. Screening of 20 CHOK1 clones stably expressing KCNQ2/3 was performed on the IonWorks HT until the best clone (judged from seal rate, current level, and stability) was obtained. The KCNQ2/3 current amplitude in the cells was found to increase from 60 ± 15 pA to 473 ± 80 pA (at –10 mV), and the expression rate was increased by 56% when the cells were incubated at 27 °C overnight. The clone used for compound screening had a seal rate of greater than 90% and an overall success rate of greater than 70%. The voltage step protocol (hold cells at –80 mV and depolarize to –10 mV for 1 s) was designed to provide moderate current but still allow for pharmacological current enhancement. EC 50 s were generated from 8-point concentration-response curves with a control compound on each plate using compounds that were also tested with conventional patch clamp. The authors found that there was a very good correlation (R 2 > 0.9) between the 2 Assays, thus demonstrating the highly predictive nature of the IonWorks Assay. (Journal of Biomolecular Screening 2007;1059-1067)

Flora Jow - One of the best experts on this subject based on the ideXlab platform.

  • validation of a medium throughput Electrophysiological Assay for kcnq2 3 channel enhancers using ionworks ht
    Journal of Biomolecular Screening, 2007
    Co-Authors: Flora Jow, Ru Shen, Pranab K. Chanda, Eugene Tseng, Howard Zhang, Jeffrey D. Kennedy, John Dunlop, Mark R. Bowlby
    Abstract:

    Enhancers of KCNQ channels are known to be effective in chronic pain models. To discover novel enhancers of KCNQ channels, the authors developed a medium-throughput Electrophysiological Assay by using the IonWorks platform. Screening of 20 CHOK1 clones stably expressing KCNQ2/3 was performed on the IonWorks HT until the best clone (judged from seal rate, current level, and stability) was obtained. The KCNQ2/3 current amplitude in the cells was found to increase from 60 ± 15 pA to 473 ± 80 pA (at –10 mV), and the expression rate was increased by 56% when the cells were incubated at 27 °C overnight. The clone used for compound screening had a seal rate of greater than 90% and an overall success rate of greater than 70%. The voltage step protocol (hold cells at –80 mV and depolarize to –10 mV for 1 s) was designed to provide moderate current but still allow for pharmacological current enhancement. EC 50 s were generated from 8-point concentration-response curves with a control compound on each plate using compounds that were also tested with conventional patch clamp. The authors found that there was a very good correlation (R 2 > 0.9) between the 2 Assays, thus demonstrating the highly predictive nature of the IonWorks Assay. (Journal of Biomolecular Screening 2007;1059-1067)

  • Validation of a Medium-Throughput Electrophysiological Assay for KCNQ2/3 Channel Enhancers Using IonWorks HT
    Journal of biomolecular screening, 2007
    Co-Authors: Flora Jow, Ru Shen, Pranab K. Chanda, Eugene Tseng, Howard Zhang, Jeffrey D. Kennedy, John Dunlop, Mark R. Bowlby
    Abstract:

    Enhancers of KCNQ channels are known to be effective in chronic pain models. To discover novel enhancers of KCNQ channels, the authors developed a medium-throughput Electrophysiological Assay by using the IonWorks platform. Screening of 20 CHOK1 clones stably expressing KCNQ2/3 was performed on the IonWorks HT until the best clone (judged from seal rate, current level, and stability) was obtained. The KCNQ2/3 current amplitude in the cells was found to increase from 60 ± 15 pA to 473 ± 80 pA (at –10 mV), and the expression rate was increased by 56% when the cells were incubated at 27 °C overnight. The clone used for compound screening had a seal rate of greater than 90% and an overall success rate of greater than 70%. The voltage step protocol (hold cells at –80 mV and depolarize to –10 mV for 1 s) was designed to provide moderate current but still allow for pharmacological current enhancement. EC 50 s were generated from 8-point concentration-response curves with a control compound on each plate using compounds that were also tested with conventional patch clamp. The authors found that there was a very good correlation (R 2 > 0.9) between the 2 Assays, thus demonstrating the highly predictive nature of the IonWorks Assay. (Journal of Biomolecular Screening 2007;1059-1067)

Michael Gurevitz - One of the best experts on this subject based on the ideXlab platform.

  • partial agonist and antagonist activities of a mutant scorpion β toxin on sodium channels
    Journal of Biological Chemistry, 2010
    Co-Authors: Izhar Karbat, Nitza Ilan, Joel Zhongli Zhang, Lior Cohen, Roy Kahn, Morris Benveniste, Todd Scheuer, William A Catterall, Dalia Gordon, Michael Gurevitz
    Abstract:

    Scorpion β-toxin 4 from Centruroides suffusus suffusus (Css4) enhances the activation of voltage-gated sodium channels through a voltage sensor trapping mechanism by binding the activated state of the voltage sensor in domain II and stabilizing it in its activated conformation. Here we describe the antagonist and partial agonist properties of a mutant derivative of this toxin. Substitution of seven different amino acid residues for Glu(15) in Css4 yielded toxin derivatives with both increased and decreased affinities for binding to neurotoxin receptor site 4 on sodium channels. Css4(E15R) is unique among this set of mutants in that it retained nearly normal binding affinity but lost its functional activity for modification of sodium channel gating in our standard Electrophysiological Assay for voltage sensor trapping. More detailed analysis of the functional effects of Css4(E15R) revealed weak voltage sensor trapping activity, which was very rapidly reversed upon repolarization and therefore was not observed in our standard Assay of toxin effects. This partial agonist activity of Css4(E15R) is observed clearly in voltage sensor trapping Assays with brief (5 ms) repolarization between the conditioning prepulse and the test pulse. The effects of Css4(E15R) are fit well by a three-step model of toxin action involving concentration-dependent toxin binding to its receptor site followed by depolarization-dependent activation of the voltage sensor and subsequent voltage sensor trapping. Because it is a partial agonist with much reduced efficacy for voltage sensor trapping, Css4(E15R) can antagonize the effects of wild-type Css4 on sodium channel activation and can prevent paralysis by Css4 when injected into mice. Our results define the first partial agonist and antagonist activities for scorpion toxins and open new avenues of research toward better understanding of the structure-function relationships for toxin action on sodium channel voltage sensors and toward potential toxin-based therapeutics to prevent lethality from scorpion envenomation.