scholarly journals Complete Cysteine-scanning Mutagenesis and Site-directed Chemical Modification of the Tn10-encoded Metal-Tetracycline/H+Antiporter

2001 ◽  
Vol 276 (23) ◽  
pp. 20330-20339 ◽  
Author(s):  
Norihisa Tamura ◽  
Satoko Konishi ◽  
Shinobu Iwaki ◽  
Tomomi Kimura-Someya ◽  
Shigeyuki Nada ◽  
...  
Keyword(s):  
Chemical Modification ◽  
Cysteine Scanning ◽  
Cysteine Scanning Mutagenesis

scholarly journals Glutathione release through connexin hemichannels: Implications for chemical modification of pores permeable to large molecules

2015 ◽  
Vol 146 (3) ◽  
pp. 245-254 ◽  
Author(s):  
Xuhui Tong ◽  
William Lopez ◽  
Jayalakshmi Ramachandran ◽  
Wafaa A. Ayad ◽  
Yu Liu ◽  
...  
Keyword(s):  
Chemical Modification ◽  
Expression System ◽  
Buthionine Sulfoximine ◽  
Reducing Agents ◽  
Xenopus Laevis Oocyte ◽  
Large Molecules ◽  
Cysteine Scanning ◽  
Cytosolic Glutathione ◽  
Cysteine Scanning Mutagenesis ◽  
Pore Lining

Cysteine-scanning mutagenesis combined with thiol reagent modification is a powerful method with which to define the pore-lining elements of channels and the changes in structure that accompany channel gating. Using the Xenopus laevis oocyte expression system and two-electrode voltage clamp, we performed cysteine-scanning mutagenesis of several pore-lining residues of connexin 26 (Cx26) hemichannels, followed by chemical modification using a methanethiosulfonate (MTS) reagent, to help identify the position of the gate. Unexpectedly, we observed that the effect of MTS modification on the currents was reversed within minutes of washout. Such a reversal should not occur unless reducing agents, which can break the disulfide thiol–MTS linkage, have access to the site of modification. Given the permeability to large metabolites of connexin channels, we tested whether cytosolic glutathione (GSH), the primary cell reducing agent, was reaching the modified sites through the connexin pore. Inhibition of gamma-glutamylcysteine synthetase by buthionine sulfoximine decreased the cytosolic GSH concentration in Xenopus oocytes and reduced reversibility of MTS modification, as did acute treatment with tert-butyl hydroperoxide, which oxidizes GSH. Cysteine modification based on thioether linkages (e.g., maleimides) cannot be reversed by reducing agents and did not reverse with washout. Using reconstituted hemichannels in a liposome-based transport-specific fractionation assay, we confirmed that homomeric Cx26 and Cx32 and heteromeric Cx26/Cx32 are permeable to GSH and other endogenous reductants. These results show that, for wide pores, accessibility of cytosolic reductants can lead to reversal of MTS-based thiol modifications. This potential for reversibility of thiol modification applies to on-cell accessibility studies of connexin channels and other channels that are permeable to large molecules, such as pannexin, CALHM, and VRAC.

scholarly journals Transmembrane IV of the high-affinity sodium-glucose cotransporter participates in sugar binding

2008 ◽  
Vol 295 (1) ◽  
pp. C64-C72 ◽  
Author(s):  
Tiemin Liu ◽  
Bryan Lo ◽  
Pam Speight ◽  
Mel Silverman
Keyword(s):  
Chemical Modification ◽  
Glucose Transporter ◽  
Xenopus Laevis Oocytes ◽  
Missense Mutations ◽  
Sugar Binding ◽  
Sodium Glucose Cotransporter ◽  
Cysteine Scanning ◽  
Electrode Voltage ◽  
Cysteine Scanning Mutagenesis ◽  
Positively Charged

Investigation of the structure/function relationships of the sodium-glucose transporter (SGLT1) is crucial to understanding the cotransporter mechanism. In the present study, we used cysteine-scanning mutagenesis and chemical modification by methanethiosulfonate (MTS) derivatives to test whether predicted transmembrane IV participates in sugar binding. Five charged and polar residues (K139, Q142, T156, K157, and D161) and two glucose/galactose malabsorption missense mutations (I147 and S159) were replaced with cysteine. Mutants I147C, T156C, and K157C exhibited sufficient expression to be studied in detail using the two-electrode voltage-clamp method in Xenopus laevis oocytes and COS-7 cells. I147C was similar in function to wild-type and was not studied further. Mutation of lysine-157 to cysteine (K157C) causes loss of phloridzin and α-methyl-d-glucopyranoside (αMG) binding. These functions are restored by chemical modification with positively charged (2-aminoethyl) methanethiosulfonate hydrobromide (MTSEA). Mutation of threonine-156 to cysteine (T156C) reduces the affinity of αMG and phloridzin for T156C by ∼5-fold and ∼20-fold, respectively. In addition, phloridzin protects cysteine-156 in T156C from alkylation by MTSEA. Therefore, the presence of a positive charge or a polar residue at 157 and 156, respectively, affects sugar binding and sugar-induced Na+ currents.

scholarly journals The Molecular Neighborhood of Subunit 8 of Yeast Mitochondrial F1F0-ATP Synthase Probed by Cysteine Scanning Mutagenesis and Chemical Modification

2003 ◽  
Vol 278 (20) ◽  
pp. 17867-17875 ◽  
Author(s):  
Andrew N. Stephens ◽  
Muhammad A. Khan ◽  
Xavier Roucou ◽  
Phillip Nagley ◽  
Rodney J. Devenish
Keyword(s):  
Chemical Modification ◽  
Atp Synthase ◽  
Cysteine Scanning ◽  
F1f0 Atp Synthase ◽  
Cysteine Scanning Mutagenesis

scholarly journals Cysteine-scanning mutagenesis around transmembrane segment VI of Tn10-encoded metal-tetracycline/H+ antiporter

FEBS Letters ◽  
1999 ◽  
Vol 461 (3) ◽  
pp. 315-318 ◽  
Author(s):  
Satoko Konishi ◽  
Shinobu Iwaki ◽  
Tomomi Kimura-Someya ◽  
Akihito Yamaguchi
Keyword(s):  
Transmembrane Segment ◽  
Cysteine Scanning ◽  
Cysteine Scanning Mutagenesis

Cysteine-Scanning Mutagenesis of the Fifth External Loop of Serotonin Transporter†

Biochemistry ◽  
2004 ◽  
Vol 43 (26) ◽  
pp. 8510-8516 ◽  
Author(s):  
Preston C. Keller ◽  
Megan Stephan ◽  
Hanna Glomska ◽  
Gary Rudnick
Keyword(s):  
Serotonin Transporter ◽  
External Loop ◽  
Cysteine Scanning ◽  
Cysteine Scanning Mutagenesis
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