ABCMdb

PMID: 8930836

Linsdell P, Hanrahan JW
Disulphonic stilbene block of cystic fibrosis transmembrane conductance regulator Cl- channels expressed in a mammalian cell line and its regulation by a critical pore residue.
J Physiol. 1996 Nov 1;496 ( Pt 3):687-93., [PubMed]

Sentences

No. Mutations Sentence Comment

14 ABCC7 p.Arg347Asp METHODS Preparation and culture of cells Experiments were carried out on baby hamster kidney (BHK) cells stably expressing either wild-type or R347D CFTR. Login to comment 16 ABCC7 p.Arg347Asp R347D pNUT-CFTR DNA (provided by Dr J. M. Rommens, Hospital for Sick Children, Toronto, Canada) was transfected into subconfluent BHK cells using Lipofectamine reagent (Life Technologies, Burlington, Canada) according to the manufacturer's directions. Login to comment 65 ABCC7 p.Arg347Asp Effect of blockers on R347D-CFTR Previous studies have identified arginine 347 in the sixth membrane-spanning region of CFTR as contributing to an important anion-binding site close to the cytoplasmic end of the Cl- channel pore (Tabcharani et al. 1993). Login to comment 66 ABCC7 p.Arg347Asp Mutation of this positively charged residue to a negatively charged aspartate (the R347D mutant) reduces channel Cl- conductance, eliminates channel block by thiocyanate A B 1.0 0-8 0-6 0-4 0-2 0-0 200 1M DNDS -- i (pA) 200 FM DNDS 1*0 - 0-8 - 0-6 - 0-4 - 0-2 - -50 V (mV) (SCN-) ions, and abolishes anomalous mole fraction behaviour seen in Cl--SCN- mixtures (Tabeharani et al. 1993). Login to comment 67 ABCC7 p.Arg347Asp We examined the effects of 200 FM internal DNDS or DIDS on macroscopic currents carried by R347D-CFTR channels stably expressed in BHK cells (Fig. 3A). Login to comment 69 ABCC7 p.Arg347Asp ABCC7 p.Arg347Asp The effect of the R347D mutation on block by DNDS and DIDS is illustrated further in Fig. 3B, which shows mean i/iO in the presence of 200 /IM DNDS or DIDS as a function of membrane potential for both wild-type and R347D-CFTR. Login to comment 72 ABCC7 p.Arg347Asp ABCC7 p.Arg347Asp Effects of intracellular DNDS and DIDS on macroscopic R347D-CFTR C1- currents A, macroscopic R347D current-voltage relationships before (0) and after (0) addition of 200 FM DNDS (left) or 200 jM DIDS (right). Login to comment 73 ABCC7 p.Arg347Asp B, comparison of the effects of 200 FM DNDS (left) or 200 uM DIDS (right) on wild-type (0) and R347D currents (0). Login to comment 78 ABCC7 p.Arg347Asp ABCC7 p.Arg347Asp Mean parameters of block of wild-type and R347D-CFTR by 200 FM intracellular DNDS or DIDS DNDS DIDS Kd(O) z Kd(O) z (/SM) (/tM) Wild-type 162 + 16 (7) 34 + 3 (7) 77 + 15 (4) 16 + 2 (4) R347D 1382 + 267 (7)** 8 + 5 (7)** 260 + 71 (6)* 15 ± 2 (6) Values for Kd(O) and z' were calculated from each experiment using eqn (2). Login to comment 83 ABCC7 p.Arg347Asp R347D-CFTR had a significantly lower affinity than wild-type for both DNDS and DIDS, with a greater than 8-fold increase in Kd(O) for DNDS and a greater than 3-fold increase in Kd(O) for DIDS observed in this mutant. Login to comment 84 ABCC7 p.Arg347Asp ABCC7 p.Arg347Asp DNDS block of R347D-CFTR was only very weakly voltage dependent, significantly less so than for wild-type, whereas DIDS block showed a similar voltage dependence for both wild-type and R347D. Login to comment 102 ABCC7 p.Arg347Cys ABCC7 p.Arg352Cys Since MTSET reacts covalently with cysteine residues, a very low rate of MTSET permeation would presumably be sufficient to block cysteine-substitutecl forms of CFTR such as R347C and R352C, whereas a similarly low rate of permeation byv a reversible blocker such as gluconate may not affect Cl- permeation. Login to comment 105 ABCC7 p.Arg347Asp The dramatic reduction in affinity for both DNDS and DIDS in the pore mutant R347D-CFTR suggests that the positively charged residue arginine 347 contributes to the binding site for both these molecules in the pore. Login to comment 113 ABCC7 p.Arg347Asp Although arginine 347 appears to contribute to the binding site for both DNDS and DIDS, it is possible that DIDS (but not DNDS) is also able to interact with another site on the channel, modulating its blocking effects on wild-type CFTR and allowing some residual blocking action on R347D-CFTR. Login to comment 191 ABCC7 p.Arg347Asp ABCC7 p.Arg347Asp Acknowledgements WAe would like to thiank Shu-Xian Zheng for constructing the R347D-CFTR BHK cell line, Jie Liao for maintaining the cell cultures and Dr Johanna Rommens for providing R347D CFTR DNA. Login to comment