scholarly journals Functional characterization of a ClC transporter by solid-supported membrane electrophysiology

2013 ◽  
Vol 141 (4) ◽  
pp. 479-491 ◽  
Author(s):  
Juan Garcia-Celma ◽  
Adrian Szydelko ◽  
Raimund Dutzler
Keyword(s):  
Chloride Channels ◽  
Ion Selectivity ◽  
Ph Dependence ◽  
Functional Characterization ◽  
Ion Concentration ◽  
Measured Signal ◽  
Supported Membrane ◽  
Experimental Limitation ◽  
Quantitative Basis ◽  
Equivalent Residue

EcClC, a prokaryotic member of the ClC family of chloride channels and transporters, works as coupled H+/Cl− exchanger. With a known structure and the possibility of investigating its behavior with different biochemical and biophysical techniques, the protein has become an important model system for the family. Although many aspects of its function have been previously characterized, it was difficult to measure transport on the same sample under different environmental conditions. To overcome this experimental limitation, we have studied EcClC by solid-supported membrane electrophysiology. The large transport-related transient currents and a simple way of relating transport rates to the measured signal have allowed a thorough investigation of ion selectivity, inhibition, and the dependence of transport on changes in ion concentration and pH. Our results confirm that the protein transports larger anions with about similar rates, whereas the smaller fluoride is not a substrate. We also show that 4,4′-diisothiocyano-2,2’-stilbenedisulfonic acid (DIDS), a known inhibitor of other anion transport protein, irreversibly inhibits EcClC from the intracellular side. The chloride dependence shows an apparent saturation at millimolar concentrations that resembles a similar behavior in eukaryotic ClC channels. Our experiments have also allowed us to quantify the pH dependence of transport. EcClC shows a strong activation at low pH with an apparent pKa of 4.6. The pronounced pH dependence is lost by the mutation of a conserved glutamate facing the extracellular solution that was previously shown to be an acceptor for transported protons, whereas it is largely retained by the mutation of an equivalent residue at the intracellular side. Our results have provided a quantitative basis for the transport behavior of EcClC, and they will serve as a reference for future investigations of novel electrogenic transporters with still-uncharacterized properties.

scholarly journals Functional Characterization of Bacterial NCx by Surface Supported Membrane Technology

2014 ◽  
Vol 106 (2) ◽  
pp. 581a
Author(s):  
Maria Barthmes ◽  
Jun Liao ◽  
Christian Wahl-Schott ◽  
Youxing Jiang ◽  
Andrea Brüggemann
Keyword(s):  
Functional Characterization ◽  
Membrane Technology ◽  
Supported Membrane

Functional characterization of an adhesive component from the embryonic chick neural retina

1979 ◽  
Vol 36 (1) ◽  
pp. 323-342
Author(s):  
R. Rutz ◽  
J. Lilien
Keyword(s):  
Conditioned Medium ◽  
Ph Dependence ◽  
Functional Characterization ◽  
Surface Binding ◽  
Cell Agglutination ◽  
Tissue Specific ◽  
A Cell ◽  
Surface Ligand ◽  
Fixed Cell

We have developed a quantitative assay for tissue-specific adhesive components which is based on the agglutination of glutaraldehyde-fixed cells. At least 2 components are required for fixed-cell agglutination: a cell-surface ligand which is obtained from tissue culture-conditioned medium, and a soluble ‘agglutinin’ which accumulates in conditioned medium from monolayer cultures. Our results suggest that the surface-binding ligand and the agglutinin interact directly, resulting in tissue-specific agglutination of cells. The agglutination reaction exhibits divalent cation, temperature, and pH dependence. Several models of cell adhesion are described; the simplest of these which can account for the data is a multicomponent model in which the 2 adhesive components have structural roles.

scholarly journals A Study into the Localized Corrosion of Magnesium Alloy Magnox Al-80

CORROSION ◽  
10.5006/3574 ◽  
2020 ◽  
Author(s):  
Ronald Clark ◽  
James Humpage ◽  
Robert Burrows ◽  
Hugh Godfrey ◽  
Mustufa Sagir ◽  
...  
Keyword(s):  
Passive Film ◽  
Ph Dependence ◽  
Chloride Concentration ◽  
Ion Concentration ◽  
Localized Corrosion ◽  
High Concentration ◽  
Transfer Resistance ◽  
High Ph ◽  
Intact Surface ◽  
Chloride Concentrations

Magnesium (Mg) non-oxidizing alloy, known as Magnox, was historically used as a fuel cladding material for the first-generation of carbon dioxide (CO<sub>2</sub>) gas-cooled nuclear reactors in the UK. Waste Magnox is currently stored in cooling ponds, pending final disposal. The corrosion resistance of Mg and its alloys is relatively poor, compared to modern cladding materials such as zirconium (Zr) alloys, so it is important to have a knowledge of the chloride concentration/pH dependence on breakdown and localized corrosion characteristics prior to waste retrievals taking place. Our results show that Magnox exhibits passivity in high pH solutions, with charge transfer resistance and passive film thicknesses showing an increase with immersion time. When chloride is added to the system the higher pH maintains Magnox passivity, as shown through a combination of potentiodynamic and time-lapse/post corrosion imaging experiments. Potentiodynamic polarization of Magnox reveals a -229 mV<sup>-decade</sup> linear dependence of breakdown potential with chloride ion concentration. The use of the scanning vibrating electrode technique (SVET) enabled the localized corrosion characteristics to be followed. At high pH where Magnox is passive, at low chloride concentrations, the anodes which form predominantly couple to the visually intact surface in the vicinity of the anode. The high pH however means that visually intact Magnox in the vicinity of the anode is less prone to breakdown, restricting anode propagation such that they remain largely static. In high chloride concentrations the higher conductivity means that the anode and cathode can couple over greater distances and so propagation along the surface can occur at a much faster rate, with the visually intact surface acting as a distributed cathode. In addition, the chloride anion itself, when present at high concentration will play a role in rapid passive film dissolution, enabling rapid anode propagation.

Abstract 17301: Functional Characterization of KCNK3 Mutants Associated With Pulmonary Arterial Hypertension Under Physiologically Relevant Heterozygous Conditions

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Michael S Bohnen ◽  
Danilo Roman-Campos ◽  
Cecile Terrenoire ◽  
Jack Jnani ◽  
Lei Chen ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Pulmonary Arterial Hypertension ◽  
Arterial Hypertension ◽  
Ph Dependence ◽  
Functional Characterization ◽  
Resting Membrane Potential ◽  
Channel Activity ◽  
Loss Of Function ◽  
Pulmonary Arterial ◽  
Specific Impact

KCNK3 encodes a two-pore domain K+ channel, TASK-1, which is inhibited by extracellular acidity and hypoxia. Expressed in a variety of tissues, including human pulmonary artery smooth muscle cells (hPASMCs), the central nervous system, pancreas, and adrenal glands, TASK-1 contributes to the resting membrane potential of cells in which it is expressed. Recently, our group reported mutations in KCNK3 underlying idiopathic pulmonary arterial hypertension (PAH), resulting from loss of TASK-1 function, partially pharmacologically rescuable with ONO-RS-082. TASK-1 dimerizes in vivo, forming functional channels with another TASK-1 subunit or with the related TASK-3 channel. TASK-1 and TASK-3 often are expressed in the same cells, although it has been reported that TASK-1 alone is expressed in the lung. Our initial study examined mutant and wildtype (WT) homodimeric TASK-1 channels expressed heterologously in COS-7 cells. Here we further characterize PAH-linked TASK-1 mutations in physiologically relevant heterozygous conditions in COS-7 and hPASMC cell lines. We engineered heterodimeric channels consisting of one mutant and one WT subunit; compared this with co-expression of mutant and WT channels; and measured channel activity with whole cell patch clamp procedures. We found a mutation specific impact of heterozygosity on channel activity. One mutation, V221L, produces a shift in pH dependence accounting for loss of function at physiological pH 7.4, partially rescued by dimerization with a WT subunit, while another, G203D, produces near complete loss of function as a homo- or hetero-dimer. The presence of TASK-3 results in greater rescue of V221L TASK-1 activity at pH 7.4 than does WT TASK-1. Additionally, under current clamp we found that ONO-RS-082 hyperpolarizes the membrane potential in hPASMCs expressing WT or V221L TASK-1, reversible by selective block of TASK-1 with ML365. Together, our results suggest (a) TASK-1 mutant heterodimers exhibit loss of function with mutation specific severity; (b) TASK-3 may rescue mutant TASK-1 and underlie a tissue specific impact of the TASK-1 mutations observed clinically; and (c) PAH TASK-1 mutants can be pharmacologically modulated in hPASMCs and alter the critically important resting membrane potential.

scholarly journals Location of Release Sites and Calcium-Activated Chloride Channels Relative to Calcium Channels at the Photoreceptor Ribbon Synapse

2011 ◽  
Vol 105 (1) ◽  
pp. 321-335 ◽  
Author(s):  
A. J. Mercer ◽  
K. Rabl ◽  
G. E. Riccardi ◽  
N. C. Brecha ◽  
S. L. Stella ◽  
...  
Keyword(s):  
Chloride Channels ◽  
Flash Photolysis ◽  
Ion Concentration ◽  
Ca Channel ◽  
Ca Channels ◽  
Vesicle Release ◽  
Voltage Range ◽  
Synaptic Release ◽  
Ribbon Synapses ◽  
Ca Currents

Vesicle release from photoreceptor ribbon synapses is regulated by L-type Ca2+ channels, which are in turn regulated by Cl− moving through calcium-activated chloride [Cl(Ca)] channels. We assessed the proximity of Ca2+ channels to release sites and Cl(Ca) channels in synaptic terminals of salamander photoreceptors by comparing fast (BAPTA) and slow (EGTA) intracellular Ca2+ buffers. BAPTA did not fully block synaptic release, indicating some release sites are <100 nm from Ca2+ channels. Comparing Cl(Ca) currents with predicted Ca2+ diffusion profiles suggested that Cl(Ca) and Ca2+ channels average a few hundred nanometers apart, but the inability of BAPTA to block Cl(Ca) currents completely suggested some channels are much closer together. Diffuse immunolabeling of terminals with an antibody to the putative Cl(Ca) channel TMEM16A supports the idea that Cl(Ca) channels are dispersed throughout the presynaptic terminal, in contrast with clustering of Ca2+ channels near ribbons. Cl(Ca) currents evoked by intracellular calcium ion concentration ([Ca2+]i) elevation through flash photolysis of DM-nitrophen exhibited EC50 values of 556 and 377 nM with Hill slopes of 1.8 and 2.4 in rods and cones, respectively. These relationships were used to estimate average submembrane [Ca2+]i in photoreceptor terminals. Consistent with control of exocytosis by [Ca2+] nanodomains near Ca2+ channels, average submembrane [Ca2+]i remained below the vesicle release threshold (∼400 nM) over much of the physiological voltage range for cones. Positioning Ca2+ channels near release sites may improve fidelity in converting voltage changes to synaptic release. A diffuse distribution of Cl(Ca) channels may allow Ca2+ influx at one site to influence relatively distant Ca2+ channels.

scholarly journals Catalysis of potato epoxide hydrolase, StEH1

2005 ◽  
Vol 390 (2) ◽  
pp. 633-640 ◽  
Author(s):  
Lisa T. Elfström ◽  
Mikael Widersten
Keyword(s):  
Steady State ◽  
Epoxide Hydrolase ◽  
Ph Dependence ◽  
Functional Characterization ◽  
Kinetic Mechanism ◽  
Kinetic Measurements ◽  
Steady State Kinetics ◽  
Substrate Conversion ◽  
Km Value ◽  
The Individual

The kinetic mechanism of epoxide hydrolase (EC 3.3.2.3) from potato, StEH1 (Solanum tuberosum epoxide hydrolase 1), was studied by presteady-state and steady-state kinetics as well as by pH dependence of activity. The specific activities towards the different enantiomers of TSO (trans-stilbene oxide) as substrate were 43 and 3 μmol·min−1·mg−1 with the R,R- or S,S-isomers respectively. The enzyme was, however, enantioselective in favour of the S,S enantiomer due to a lower Km value. The pH dependences of kcat with R,R or S,S-TSO were also distinct and supposedly reflecting the pH dependences of the individual kinetic rates during substrate conversion. The rate-limiting step for TSO and cis- and trans-epoxystearate was shown by rapid kinetic measurements to be the hydrolysis of the alkylenzyme intermediate. Functional characterization of point mutants verified residues Asp105, Tyr154, Tyr235 and His300 as crucial for catalytic activity. All mutants displayed drastically decreased enzymatic activities during steady state. Presteady-state measurements revealed the base-deficient H300N (His300→Asn) mutant to possess greatly reduced efficiencies in catalysis of both chemical steps (alkylation and hydrolysis).

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