Cystic fibrosis transmembrane conductance regulator: using differential reactivity toward channel-permeant and channel-impermeant thiol-reactive probes to test a molecular model for the pore

Abstract

The sixth transmembrane segment (TM6) of the CFTR chloride channel has been intensively investigated. The effects of amino acid substitutions and chemical modification of engineered cysteines (cysteine scanning) on channel properties strongly suggest that TM6 is a key component of the anion-conducting pore, but previous cysteine-scanning studies of TM6 have produced conflicting results. Our aim was to resolve these conflicts by combining a screening strategy based on multiple, thiol-directed probes with molecular modeling of the pore. CFTR constructs were screened for reactivity toward both channel-permeant and channel-impermeant thiol-directed reagents, and patterns of reactivity in TM6 were mapped onto two new, molecular models of the CFTR pore: one based on homology modeling using Sav1866 as the template and a second derived from the first by molecular dynamics simulation. Comparison of the pattern of cysteine reactivity with model predictions suggests that nonreactive sites are those where the TM6 side chains are occluded by other TMs. Reactive sites, in contrast, are generally situated such that the respective amino acid side chains either project into the predicted pore or lie within a predicted extracellular loop. Sites where engineered cysteines react with both channel-permeant and channel-impermeant probes occupy the outermost extent of TM6 or the predicted TM5-6 loop. Sites where cysteine reactivity is limited to channel-permeant probes occupy more cytoplasmic locations. The results provide an initial validation of two, new molecular models for CFTR and suggest that molecular dynamics simulation will be a useful tool for unraveling the structural basis of anion conduction by CFTR.

Figure 1

Differential effects of [Au(CN)₂]⁻ and [Ag(CN)₂]⁻ on Cys-less CFTR. Exposure to [Au(CN)₂]⁻ (1 mM) produced a “lyotropic” block that was not altered by exposure to KCN (1 mM). Exposure to [Ag(CN)₂]⁻ (1 mM) produced a substantially smaller block that was exacerbated by adding KCN in the continued presence of [Ag(CN)₂]⁻.

Figure 2

Functional impact of covalent labeling of L333C CFTR was charge-independent. After activation by isoproterenol and IBMX, exposure to either 1 mM MTSET⁺ or 1 mM MTSES⁻ produced a substantial inhibition of conductance.

Figure 3

Selective reactivity of F337C CFTR. Exposure of an oocyte to 1 mM MTSET⁺ or 1 mM MTSES⁻ resulted in small, reversible reductions of conductance. In some experiments, no change in conductance was seen after exposure to these reagents. Subsequent exposure of the oocyte to [Au(CN)₂]⁻ (1 mM) produced a profound inhibition of conductance that was only slightly reversed by removing [Au(CN)₂]⁻ from the perfusate. Inhibition was not reversed by 1 mM 2-ME but was reversed by exposure to 1 mM KCN.

Figure 4

Differential reactivity of S341C/Cys-less CFTR toward [Ag(CN)₂]⁻ and [Au(CN)₂]⁻. Inhibition of conductance by [Ag(CN)₂]⁻ (100 μM) was reversed by adding KCN in the continued presence of [Ag(CN)₂]⁻, indicative of a ligand exchange or ligand addition reaction. In contrast, inhibition by [Au(CN)₂]⁻ (1 mM) was unaffected by adding KCN as expected for lyotropic block.

Figure 5

R352C/Cys-less CFTR was reactive toward [Ag(CN)₂]⁻ (1 mM) as judged by reversal of inhibition by adding KCN (1 mM) in the continued presence of [Ag(CN)₂]⁻.

Figure 6

Three views of two models of the CFTR pore based on Sav1866 and a 5 ns molecular dynamics simulation. Insets show the portion of the protein that is enlarged in each panel, and dashed lines indicate the approximate boundaries of the lipid bilayer. Also, the contains two movies that offer an overall view of the two models. Cytoplasmic domains have been removed from the enlarged views. (A) Top view looking “down” into the pore from the extracellular side. (B) Side view with TMs 9, 10, and 12 partially removed to reveal the “pore-lining” face of TM6. (C) Side view from behind the TM3−4 loop. Residues indicated in black are those where substituted cysteines were unreactive with any of the probes employed in this study. Residues colored in red and pink represent sites where thiol reactivity was detected toward both channel-permeant and channel-impermeant, thiol-directed reagents. The pink residues are sites where the resulting functional effects were charge-independent, whereas those in red represent sites where functional effects were distinctly charge-dependent. Residues colored in yellow and orange represent those sites where cysteines reacted exclusively with permeant reagents, [Ag(CN)₂]⁻ and [Au(CN)₂]⁻. [Ag(CN)₂]⁻ reacted at all of these sites while [Au(CN)₂]⁻ reacted only at those labeled in orange. All molecular representations were generated using “Visual Molecular Dynamics” (vmd) (54).

Figure 7

Views of R352 and its relation to D993 in the 1 and 5 ns molecular models. Atom coloring is carbon = cyan, nitrogen = blue, white = hydrogen, and red = oxygen. (A) View from the top of a cross section of the pore (0 ns model) showing that both R352 and D993 side chains are predicted to project into the pore. (B, C) Side views showing the change in relative position of the two residues in the 0 and 5 ns models. It can be seen that the nitrogen on the arginine side chain and the oxygen on the aspartate side chain move closer in the 5 ns model.

Figure 8

Summary of the results of previous cysteine-scanning studies of TM6 of the CFTR chloride channel compared to the present study. Note the lack of consistent results reported for F337C, S341C, I344C, R347C, T351C, R352C, and Q353C (shaded). ● = sites where reactivity with channel-impermeant probes was inferred. ◆ = sites where reactivity with [Ag(CN)₂]⁻ was inferred. ◇ = sites where reactivity with [Au(CN)₂]⁻ was inferred. ○ = sites scored as nonreactive. Superscripts refer to bibliographic references.