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.

The CFTR Ion Channel: Gating, Regulation, and Anion Permeation

“…Selectivity arises because those anions that partition more effectively into the pore from water (e.g., SCN) also reside longer on average within the pore (bind more tightly) because of a greater difference between their interaction energies with water (hydration energies) and with the lower dielectric pore interior (solvation energies). To a first approximation this bias-type selectivity mechanism (Smith et al 1999) describes how permeant anions traverse the CFTR pore with of course the limit that some anions are too big to pass through the pore in the first place (Alexander et al 2009). …”

“…Linsdell et al (2000) first reported that mutations at residues F337 and T338 in TM6 strongly affected the permeability sequence. Those findings were extended by testing additional TM6 mutants Ge et al 2004) and by using cysteine scanning to explore the accessibility of TM6 residues (Alexander et al 2009;Bai et al 2010;El Hiani and Linsdell 2010). Alexander et al (2009) compared the effects of two types of reactive compounds on a series of TM6 cysteine mutants: large methanethiosulfonate (MTS) compounds that are not expected to traverse the pore and smaller pseudohalides that both traverse the CFTR pore and are thiol reactive (Ag(CN) 2 and Au(CN) 2 ).…”

“…Based on the ATP-bound configuration of ABC exporters, several homology models of CFTR's "open state" were composed Alexander et al 2009;Mornon et al 2009;Norimatsu et al 2012). However, these homology models, like their counterparts of ABC exporters, all lack an uninterrupted pathway for ion permeation.…”

Two Salt Bridges Differentially Contribute to the Maintenance of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channel Function

“…Dysfunction of CFTR is directly associated with three devastating diseases: cystic fibrosis, polycystic kidney disease, and secretory diarrhea. Based on functional data and homology models, CFTR has been predicted to contain five functional domains: two membranespanning domains (MSDs), each including six transmembrane (TM) helices; two nucleotide binding domains (NBD1 and NBD2); and a unique regulatory (R) domain, which carries multiple protein kinase A (PKA) consensus phosphorylation sites and is unique to CFTR in the ABC superfamily (1)(2)(3)(4)(5)(6). Functional studies from multiple groups have suggested that TM6 plays an essential role and TM12 contributes less to anion conduction and permeation properties in the CFTR channel pore (7)(8)(9)(10)(11).…”

“…The channel state of CFTR in the homology model is unclear, but it is evident that the four salt bridge amino acids sit close to each other. Alexander et al found that Arg 352 and Asp 993 start out quite a distance away from each other in their molecular dynamics simulations of CFTR conformational change and move toward to each other as the simulation progresses toward the open state (3). In the bacterial Na ϩ channel and Kv7.1 channels, it has been reported that dynamic electrostatic interactions are involved in channel gating (17)(18)(19)(20).…”