scholarly journals Development of a Colorimetric Method for Functional Chloride Channel Assay

2004 ◽  
Vol 9 (7) ◽  
pp. 607-613 ◽  
Author(s):  
Weimin Tang ◽  
Mary Jo Wildey
Keyword(s):  
Chloride Channel ◽  
High Throughput Screening ◽  
Colorimetric Detection ◽  
Colorimetric Method ◽  
Anion Channel ◽  
Aminobutyric Acid ◽  
Anion Channels ◽  
Acid Type ◽  
A Cell ◽  
Inwardly Rectifying

Anion channels play significant physiological roles in humans and animals. However, the effort of screening for anion channel modulators was limited by the available assay technologies. This report discusses the development of a cell-based functional chloride channel assay using iodine as the chloride channel functional indicator. Iodine concentrations were measured with modified Sandell-Kolthoff reaction using colorimetric detection. The assay was rapid and quantitative. When WSS-1 cells were activated by γ-aminobutyric acid (GABA) in the condition that γ-aminobutyric acid type A receptor (GABAA receptor) conducted outwardly rectifying chloride channel function, the EC50 of GABA was 7.69 μM. IC50 swere 0.53 μM for bicuculline and 3.1 μM for picrotoxin, respectively, in the presence of 10 μM GABA. When Capan-1 cells were activated by forskolin, the EC50 was 0.14 μM. The assay can also be applied to inwardly rectifying anion channels as exemplified by GABAA channel with an EC50 of 294 μM. Thus, the assay is universal and reliable and can be used for anion channel high-throughput screening.

scholarly journals Interaction of Non-competitive Blockers within the γ-Aminobutyric Acid Type A Chloride Channel Using Chemically Reactive Probes as Chemical Sensors for Cysteine Mutants

1999 ◽  
Vol 274 (36) ◽  
pp. 25350-25354 ◽  
Author(s):  
Philippe Perret ◽  
Xavier Sarda ◽  
Mark Wolff ◽  
Tai-Teh Wu ◽  
Dean Bushey ◽  
...  
Keyword(s):  
Chemical Sensors ◽  
Chloride Channel ◽  
Type A ◽  
Aminobutyric Acid ◽  
Acid Type

Maxi-channels recorded in situ from ICC and pericytes associated with the mouse myenteric plexus

2012 ◽  
Vol 302 (7) ◽  
pp. C1055-C1069 ◽  
Author(s):  
Sean P. Parsons ◽  
Wolfgang A. Kunze ◽  
Jan D. Huizinga
Keyword(s):  
Chloride Channel ◽  
Myenteric Plexus ◽  
Anion Channel ◽  
Anion Channels ◽  
Chloride Permeability ◽  
Cation Permeability ◽  
Dependent Inactivation ◽  
Method Of Measurement ◽  
Cells Of Cajal

Ion channels are fundamental to gastrointestinal pacemaking by interstitial cells of Cajal (ICC). Previously, we have recorded a high-conductance chloride channel (HCCC) from ICC, both in culture and in situ, associated with the myenteric plexus. The biophysical properties of the HCCC (conductance, subconductances, voltage- and time-dependent inactivation) suggest it is a member of a class called the maxi-anion channels. In this study we further investigated the properties of the HCCC in situ. Our main finding was that the HCCC is not strictly a chloride channel but has a relative sodium-chloride permeability (PNa/Cl) of 0.76 to 1.64 (depending on the method of measurement). Therefore, we have renamed the HCCC the “maxi-channel.” A maxi-channel was also expressed by pericytes associated with the vasculature near the myenteric plexus. This had a lower PNa/Cl (0.33 to 0.49, depending on the method of measurement) but similar conductance (326 ± 7 vs. 316 ± 24 pS for ICC). This is the first report of cation permeability equaling anion permeability in a maxi-anion channel. As such, the properties of the maxi-channels described in this article may have implications for the maxi-anion channel field, as well as for studies of their role in ICC and pericytes.

scholarly journals Human  -aminobutyric acid type B receptors are differentially expressed and regulate inwardly rectifying K+ channels

1998 ◽  
Vol 95 (25) ◽  
pp. 14991-14996 ◽  
Author(s):  
K. Kaupmann ◽  
V. Schuler ◽  
J. Mosbacher ◽  
S. Bischoff ◽  
H. Bittiger ◽  
...  
Keyword(s):  
Differentially Expressed ◽  
Aminobutyric Acid ◽  
Type B ◽  
K Channels ◽  
Acid Type ◽  
Inwardly Rectifying

Structural Comparisons of Ligand-gated Ion Channels in Open, Closed, and Desensitized States Identify a Novel Propofol-binding Site on Mammalian γ-Aminobutyric Acid Type A Receptors

2015 ◽  
Vol 122 (4) ◽  
pp. 787-794 ◽  
Author(s):  
Nicholas P. Franks
Keyword(s):  
Ion Channel ◽  
Chloride Channel ◽  
Transmembrane Domain ◽  
Type A ◽  
Transmembrane Domains ◽  
Aminobutyric Acid ◽  
State Dependent ◽  
Novel Approach ◽  
Channel Opening ◽  
Acid Type

Abstract Background: Most anesthetics, particularly intravenous agents such as propofol and etomidate, enhance the actions of the neurotransmitter γ-aminobutyric acid (GABA) at the GABA type A receptor. However, there is no agreement as where anesthetics bind to the receptor. A novel approach would be to identify regions on the receptor that are state-dependent, which would account for the ability of anesthetics to affect channel opening by binding differentially to the open and closed states. Methods: The open and closed structures of the GABA type A receptor homologues Gloeobacter ligand–gated ion channel and glutamate-gated chloride channel were compared, and regions in the channels that move on channel opening and closing were identified. Docking calculations were performed to investigate possible binding of propofol to the GABA type A β3 homomer in this region. Results: A comparison between the open and closed states of the Gloeobacter ligand–gated ion channel and glutamate-gated chloride channel channels identified a region at the top of transmembrane domains 2 and 3 that shows maximum movement when the channels transition between the open and closed states. Docking of propofol into the GABA type A β3 homomer identified two putative binding cavities in this same region, one with a high affinity and one with a lower affinity. Both cavities were adjacent to a histidine residue that has been photolabeled by a propofol analog, and both sites would be disrupted on channel closing. Conclusions: These calculations support the conclusion of a recent photolabeling study that propofol acts at a site at the interface between the extracellular and transmembrane domains, close to the top of transmembrane domain 2.

Directed Evolution of P450 Fatty Acid Decarboxylases via High-Throughput Screening Towards Improved Catalytic Activity

2019 ◽  
Author(s):  
Huifang Xu ◽  
Weinan Liang ◽  
Linlin Ning ◽  
Yuanyuan Jiang ◽  
Wenxia Yang ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Fatty Acid ◽  
Directed Evolution ◽  
High Throughput ◽  
High Throughput Screening ◽  
Colorimetric Detection ◽  
Screening Method ◽  
Random Mutagenesis ◽  
Direct Production ◽  
Consumption Activity

P450 fatty acid decarboxylases (FADCs) have recently been attracting considerable attention owing to their one-step direct production of industrially important 1-alkenes from biologically abundant feedstock free fatty acids under mild conditions. However, attempts to improve the catalytic activity of FADCs have met with little success. Protein engineering has been limited to selected residues and small mutant libraries due to lack of an effective high-throughput screening (HTS) method. Here, we devise a catalase-deficient <i>Escherichia coli</i> host strain and report an HTS approach based on colorimetric detection of H<sub>2</sub>O<sub>2</sub>-consumption activity of FADCs. Directed evolution enabled by this method has led to effective identification for the first time of improved FADC variants for medium-chain 1-alkene production from both DNA shuffling and random mutagenesis libraries. Advantageously, this screening method can be extended to other enzymes that stoichiometrically utilize H<sub>2</sub>O<sub>2</sub> as co-substrate.

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