ABCMdb

PMID: 24086355

Rahman KS, Cui G, Harvey SC, McCarty NA
Modeling the conformational changes underlying channel opening in CFTR.
PLoS One. 2013 Sep 27;8(9):e74574. doi: 10.1371/journal.pone.0074574. eCollection 2013., [PubMed]

Sentences

No. Mutations Sentence Comment

65 ABCC7 p.Arg334Cys State-Dependent Accessibility of R334C The arginine at position 334 on TM6 lies at the outer mouth of the pore, and has been suggested to play a role in attracting anions [33,34]. Login to comment 66 ABCC7 p.Arg334Cys It has been observed, however, that modification of cysteine mutants at this site (R334C) occurs only in the closed state, with the site apparently being inaccessible when the channel is open [51-53]. Login to comment 68 ABCC7 p.Arg334Cys In an open state O-CFTR model in which the arginine at position 334 was mutated to a cysteine in silico, R334C is buried by many of its neighboring residues in ECL3 and is found to have a fractional solvent-accessible surface area (SASA) of only 3.8%. Login to comment 69 ABCC7 p.Arg334Cys By comparison, the C0-CFTR model presents R334C in a relatively exposed conformation, with 32.9% of its surface area accessible to solvent. Login to comment 70 ABCC7 p.Arg334Cys This increased accessibility may translate to the higher observed rates of reaction of R334C with extracellular sulfhydryl-modifying reagents in the closed state. Login to comment 97 ABCC7 p.Arg334Cys State dependent-accessibility of R334C. Login to comment 98 ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys These images show CFTR from the extracellular side, with the mutant residue R334C represented in CPK colors, and all residues with atoms within 5 A da; of R334C shown as pink spheres. Login to comment 99 ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys R334C is largely buried by its neighboring residues in the open channel state, exposing only 3.8% of its surface area to solvent. On the other hand, in the closed channel, R334C is more exposed (32.9% accessible). Login to comment 100 ABCC7 p.Arg334Cys This is in accordance with experimental results demonstrating the preferential closed-state accessibility of R334C [51,52]. Login to comment 144 ABCC7 p.Arg334Cys ABCC7 p.Glu217Cys To provide experimental confirmation of the closed and open structures, and the transitions between the two, we asked whether cysteines engineered at these two positions in the double-mutant, R334C/E217C-CFTR, could be functionally crosslinked. Login to comment 146 ABCC7 p.Arg334Cys ABCC7 p.Glu217Cys The traces in Figure 7 show that R334C/E217C-CFTR can repeatedly be activated by stimulation of the co-expressed beta2-adrenergic receptor using isoproterenol, without substantial decrement in peak current prior to exposure to the crosslinker MTS-2-MTS. Login to comment 147 ABCC7 p.Arg334Cys ABCC7 p.Glu217Cys When the same cell was exposed to MTS-2-MTS in the absence of isoproterenol (Figure 7B), when most of the channels should be closed, subsequent exposure to isoproterenol failed to activate CFTR channels to the same degree as prior to MTS-2-MTS; these results are consistent with the notion that R334C and E217C are positioned very near each other in the channel closed state. Login to comment 152 ABCC7 p.Arg334Cys ABCC7 p.Glu217Cys In control experiments, exposure to MTS-2-MTS did not have similar effects on the single mutants R334C-CFTR and E217C-CFTR, with respect to the ability to re-open channels after MTS-2-MTS exposure (Figure S6). Login to comment 154 ABCC7 p.Arg334Cys ABCC7 p.Glu217Cys Inspection of the C0- and O-CFTR models indicates that the side chains of R334C and E217C are, indeed, close enough to be crosslinked by MTS-2-MTS only in the closed state (Figure S8). Login to comment 173 ABCC7 p.Arg334Cys ABCC7 p.Glu217Cys Crosslinking R334C to E217C locks CFTR channels into the closed state. Login to comment 176 ABCC7 p.Arg334Cys ABCC7 p.Glu217Cys B: Representative trace (left) and summary data (right) for macroscopic currents measured from R334C/E217C-CFTR with addition of the crosslinker MTS-2-MTS in the absence of isoproterenol (ISO), used to activate channels. Login to comment 178 ABCC7 p.Arg334Cys ABCC7 p.Glu217Cys C: Representative trace and summary data for currents measured from R334C/E217C-CFTR with the crosslinker MTS-2-MTS added in the continuing presence of isoproterenol. Login to comment 266 ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys ABCC7 p.Glu217Cys ABCC7 p.Glu217Cys (TIF) Figure S6 Effects of 1 mM MTS2-2MTS on R334C-CFTR and E217C-CFTR channels. Representative traces (left) and summary data (right) for macroscopic currents measured from R334C- (A) and E217C-CFTR (B) by two-electrode voltage clamp. Login to comment 270 ABCC7 p.Arg334Cys ABCC7 p.Glu217Cys MTS-2-MTS also appeared to covalently decrease macroscopic current at R334C-CFTR, but not E217C-CFTR, perhaps due to alteration of charge or side-chain volume. Login to comment 275 ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys ABCC7 p.Glu217Cys ABCC7 p.Glu217Cys (TIF) Figure S7 Effects of MTSET (ET+) and MTSES (ES-) on R334C/E217C-CFTR channels. Representative traces (left) and summary data (right) for macroscopic currents measured from R334C/E217C-CFTR by two-electrode voltage clamp with addition of the 1 mM monofunctional MTS reagents MTSET+ (ET+ ) or MTSES2 (ES2 ) in the presence of isoproterenol (ISO). Login to comment 277 ABCC7 p.Arg334Cys ABCC7 p.Glu217Cys Both ET+ and ES2 covalently bound to R334C/E217C-CFTR. Login to comment 284 ABCC7 p.Arg334Cys ABCC7 p.Glu217Cys (TIF) Figure S8 Distances between residues in R334C-E217C Double Mutant. Login to comment 410 ABCC7 p.Thr338Cys Serrano JR, Liu X, Borg ER, Alexander CS, Shaw CF, et al. (2006) CFTR: Ligand exchange between a permeant anion ([Au(CN)2]2 ) and an engineered cysteine (T338C) blocks the pore. Biophys J 91: 1737-1748. doi:10.1529/ biophysj.105.078899. Login to comment