ABCMdb

PMID: 19466983

Li MS, Demsey AF, Qi J, Linsdell P
Cysteine-independent inhibition of the CFTR chloride channel by the cysteine-reactive reagent sodium (2-sulphonatoethyl) methanethiosulphonate.
Br J Pharmacol. 2009 Jul;157(6):1065-71. Epub 2009 May 19., [PubMed]

Sentences

No. Mutations Sentence Comment

28 ABCC7 p.Val510Ala To facilitate channel protein maturation and expression in the cell membrane, the V510A mutation (Wang et al., 2007) was introduced into this construct using the QuikChange site directed mutagenesis system as described previously (Gong et al., 2002) and verified by DNA sequencing. Login to comment 29 ABCC7 p.Val510Ala Baby hamster kidney (BHK) and human embryonic kidney cells were transiently transfected with pIRES2-EGFP-cys-less/V510A CFTR cDNA as described previously (Gong et al., 2002), except that 24 h after transfection, cells were transferred to 27°C to promote mature protein expression (see Supporting Information, Figure S1). Login to comment 32 ABCC7 p.Val510Ala Once trafficked to the cell membrane (see Supporting Information, Figure S1), the properties of cys-less/V510A CFTR channel currents appeared identical to those of wild-type CFTR studied previously, with the exception that single channel conductance appeared increased (see Figures 2 and 3). Login to comment 55 ABCC7 p.Val510Ala Results Expression of cys-less/V510A CFTR in BHK cells led to the appearance of PKAand ATP-dependent PPi-stimulated macroscopic currents when the cells were grown at 27°C. Examples of such currents, which had similar properties to those of wild-type CFTR studied many times previously (e.g. Linsdell and Hanrahan, 1998; Gong et al., 2002), are shown in Figure 1. Login to comment 66 ABCC7 p.Val510Ala (A) An example of the leak-subtracted macroscopic current-voltage relationship for cys-less/V510A-CFTR following maximal current stimulation with protein kinase A catalytic subunit (PKA), adenosine 5'-triphosphate (ATP) and pyrophosphate (PPi). Login to comment 103 ABCC7 p.Arg303Cys This could be misinterpreted as being the result of charge deposition in the permeation pathway due to covalent modification of a cysteine side chain. For example, we previously showed that intracellular application of a low concentration of MTSES (200 mmol·L-1 ) led to outward rectification of the macroscopic current-voltage relationship in a CFTR mutant (R303C) but not wild-type CFTR, which we proposed was due to the electrostatic effects of the addition of a negative charge close to the inner mouth of the pore by covalent modification of the introduced cysteine side chain (St. Aubin and Linsdell 2006). Login to comment 104 ABCC7 p.Arg303Cys The change in macroscopic current-voltage relationship shape observed in MTSES-modified R303C-CFTR is, however, similar to that resulting from voltage-dependent block by MTSES (Figure 1A). Login to comment 105 ABCC7 p.Arg303Cys Nevertheless, it seems unlikely that open channel block contributed to the reported effects of MTSES on R303C-CFTR, since (i) qualitatively opposite effects were observed with positively charged MTSET, and (ii) these effects were observed at MTSES concentrations of only 200 mmol·L-1 , a concentration which does not result in significant current blockage (Figure 1). Login to comment