State-dependent Access of Anions to the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Pore

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl ؊ channel is gated by intracellular factors; however, conformational changes in the channel pore associated with channel activation have not been identified. We have used patch clamp recording to investigate the state-dependent accessibility of substituted cysteine residues in the CFTR channel pore to a range of cysteine-reactive reagents applied to the extracellular side of the membrane. Using functional modification of the channel current-voltage relationship as a marker of modification, we find that several positively charged reagents are able to penetrate deeply into the pore from the outside irrespective of whether or not the channels have been activated. In contrast, access of three anionic cysteine-reactive reagents, the methanesulfonate sodium (2-sulfonatoethyl)methanesulfonate, the organic mercurial p-chloromercuriphenylsulfonic acid, and the permeant anion Au(CN) 2 ؊ , to several different sites in the pore is strictly limited prior to channel activation. This suggests that in nonactivated channels some ion selectivity mechanism exists to exclude anions yet permit cations into the channel pore from the extracellular solution. We suggest that activation of CFTR channels involves a conformational change in the pore that removes a strong selectivity against anion entry from the extracellular solution. We propose further that this conformational change occurs in advance of channel opening, suggesting that multiple distinct closed pore conformations exist.

Dynamics Intrinsic to Cystic Fibrosis Transmembrane Conductance Regulator Function and Stability

“…The channel pore architecture has been experimentally probed using cysteine mutants and thiol-reactive reagents to determine the accessibility of particular sites in the pore. Taken together, these studies are strongly supportive of the Sav1866-based homology models (Zhang et al 2005;Fatehi and Linsdell 2008;Alexander et al 2009). They show a pore that is wide on the extracellular side and becomes more inaccessible to molecular probes, especially anionic probes, moving toward the cytoplasmic surface.…”

Locating a Plausible Binding Site for an Open-Channel Blocker, GlyH-101, in the Pore of the Cystic Fibrosis Transmembrane Conductance Regulator

“…A conformational change of this sort would be consistent with the state-dependent reactivity of the F337C and T338C CFTRs observed in the current study. The MsbAbased model described by Mornon et al (2009) also suggests that the side chain of Arg334 protrudes into the external aqueous environment; when Arg334 is mutated to a cysteine 2 The findings presented here regarding the state-dependent reactivity of engineered cysteines are at odds with previous reports (Wang and Linsdell, 2012;Fatehi and Linsdell, 2008). Wang and Linsdell (2012) Ϫ and suggested that the reaction of an engineered cysteine at position 338 with externally applied reagents was favored in the closed state.…”

“…We observed reaction rates for MTSES Ϫ with T338C/wt and T338C/E1371Q CFTRs that were similar to those observed by Beck et al (2008) (data not shown), which supports the idea that the T338C CFTR reacts in the open state. Fatehi and Linsdell (2008) reported, contrary to our findings, that reactions of MTSES Ϫ and [Au(CN) 2 ] Ϫ with a cysteine at position 334 were favored in the activated state of the channel. Cells expressing the R334C CFTR were preincubated with MTSES Ϫ with and without an activating cocktail of forskolin and IBMX, and the reaction, which was inferred on the basis of changes in the rectification ratio of the CFTR I-V curve, was observed only with cells exposed to the activating cocktail.…”

“…With respect to the state-dependent reactivity of engineered cysteines toward externally applied reagents, it was suggested that it might be possible to distinguish between the closed state of the channel before phosphorylation (the preactivation state in the current study) and the closed state that is visited transiently, after phosphorylation, during ATP-dependent channel gating (Fatehi and Linsdell, 2008). As a test of this notion, we compared the modification rates before and after activation for a cysteine substituted at position 334.…”

“…Similarly, the outcome of cell preincubation with [Au (CN) 2 ] Ϫ and the subsequent reversal of CFTR blockade by CN Ϫ can be influenced by metal contamination and the redox state of the cell (Liu, 2008). It is possible that the state dependence observed by Fatehi and Linsdell (2008) was attributable to such an artifact. with Maestro 9.1 (Schrödinger) in the MsbA-based model, the reactive thiolate is clearly accessible from the extracellular solution (Fig.…”