The cystic fibrosis transmembrane conductance regulator (CFTR) is associated with expression of a chloride conductance that is defective in cystic fibrosis (CF). Xenopus oocytes injected with RNA coding for CFTR that contained mutations in the first nucleotide binding fold (NBF1) expressed chloride currents in response to raising adenosine 3',5'-monophosphate (cAMP) with forskolin and 3-isobutyl-1-methylxanthine (IBMX). The mutant CFTRs were less sensitive than wild-type CFTR to this activating stimulus, and the reduction in sensitivity correlated with the severity of cystic fibrosis in patients carrying the corresponding mutations. This demonstration provides the basis for detailed analyses of NBF1 function and suggests potential pharmacologic treatments for cystic fibrosis.

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.

CFTR: Mechanism of Anion Conduction. Physiol. Rev. 79, Suppl.: S47-S75, 1999. - The purpose of this review is to collect together the results of recent investigations of anion conductance by the cystic fibrosis transmembrane conductance regulator along with some of the basic background that is a prerequisite for developing some physical picture of the conduction process. The review begins with an introduction to the concepts of permeability and conductance and the Nernst-Planck and rate theory models that are used to interpret these parameters. Some of the physical forces that impinge on anion conductance are considered in the context of permeability selectivity and anion binding to proteins. Probes of the conduction process are considered, particularly permeant anions that bind tightly within the pore and block anion flow. Finally, structure-function studies are reviewed in the context of some predictions for the origin of pore properties.

We developed molecular models for the CFTR chloride channel based on the prokaryotic ABC transporter, Sav1866. Here we analyze predicted pore geometry and side-chain orientations for TMs 3, 6, 9 and 12; with particular attention to the location of the rate-limiting barrier for anion conduction. Side-chain orientations assayed by cysteine scanning were found to be from 77% to 90% in accord with model predictions. The predicted geometry of the anion conduction path was defined by a space-filling model of the pore and confirmed by visualizing the distribution of water molecules from a molecular dynamics (MD) simulation. Pore shape is that of an asymmetric hour glass, comprising a shallow outward-facing vestibule that tapers rapidly toward a narrow “bottleneck” linking the outer vestibule to a large inner cavity extending toward the cytoplasmic extent of the lipid bilayer. The junction between the outer vestibule and the bottleneck features an outward–facing rim marked by T338 in TM6 and I1131 in TM12, consistent with the observation that cysteines at both of these locations reacted with both channel-permeant and channel-impermeant, thiol-directed reagents. Conversely, cysteines substituted for S341 in TM6 or T1134 in TM12, predicted by the model to lie below the rim of the bottleneck, were found to react exclusively with channel-permeant reagents applied from the extracellular side. The predicted dimensions of the bottleneck are consistent with the demonstrated permeation of Cl− pseudohalide anions, water and urea.

We compared the effects of mutations in transmembrane segments (TMs) TM1, TM5, and TM6 on the conduction and activation properties of the cystic fibrosis transmembrane conductance regulator (CFTR) to determine which functional property was most sensitive to mutations and, thereby, to develop a criterion for measuring the importance of a particular residue or TM for anion conduction or activation. Anion substitution studies provided strong evidence for the binding of permeant anions in the pore. Anion binding was highly sensitive to point mutations in TM5 and TM6. Permeability ratios, in contrast, were relatively unaffected by the same mutations, so that anion binding emerged as the conduction property most sensitive to structural changes in CFTR. The relative insensitivity of permeability ratios to CFTR mutations was in accord with the notion that anion-water interactions are important determinants of permeability selectivity. By the criterion of anion binding, TM5 and TM6 were judged to be likely to contribute to the structure of the anion-selective pore, whereas TM1 was judged to be less important. Mutations in TM5 and TM6 also dramatically reduced the sensitivity of CFTR to activation by 3-isobutyl 1-methyl xanthine (IBMX), as expected if these TMs are intimately involved in the physical process that opens and closes the channel.

The goal of the experiments described here was to explore the possible role of fixed charges in determining the conduction properties of CFTR. We focused on transmembrane segment 6 (TM6) which contains four basic residues (R334, K335, R347, and R352) that would be predicted, on the basis of their positions in the primary structure, to span TM6 from near the extracellular (R334, K335) to near the intracellular (R347, R352) end. Cysteines substituted at positions 334 and 335 were readily accessible to thiol reagents, whereas those at positions 347 and 352 were either not accessible or lacked significant functional consequences when modified. The charge at positions 334 and 335 was an important determinant of CFTR channel function. Charge changes at position 334—brought about by covalent modification of engineered cysteine residues, pH titration of cysteine and histidine residues, and amino acid substitution—produced similar effects on macroscopic conductance and the shape of the I-V plot. The effect of charge changes at position 334 on conduction properties could be described by electrodiffusion or rate-theory models in which the charge on this residue lies in an external vestibule of the pore where it functions to increase the concentration of Cl adjacent to the rate-limiting portion of the conduction path. Covalent modification of R334C CFTR increased single-channel conductance determined in detached patches, but did not alter open probability. The results are consistent with the hypothesis that in wild-type CFTR, R334 occupies a position where its charge can influence the distribution of anions near the mouth of the pore.

To investigate the functional significance of individual consensus phosphorylation sites within the R domain of cystic fibrosis transmembrane conductance regulator (CFTR), serines were eliminated by substituting them with alanine. Included in this analysis were serine-660, -670, -686, -700, -712, -737, -768, -795, and -813, which lie within protein kinase A consensus sequences, and serine-641, which does not. Elimination of single potential phosphorylation sites altered the sensitivity of CFTR (expressed in Xenopus oocytes) to activating conditions in a manner that was highly site dependent. Substitution at serine-660, -670, -700, -795, or -813 significantly increased the half-maximal activation constant (KA) for activation by 3-isobutyl-1-methylxanthine, which is consistent with the hypothesis that phosphorylation at any of these sites promotes CFTR activation. The effect of substitution at serine-813 was significantly greater than at the other sites. In contrast, alanine substitution at serine-737 or -768 actually decreased the KA for activation, suggesting that phosphorylation at either of these sites is inhibitory. Substitution at serine-641, -686, and -712 had no significant effect on activation sensitivity. The effects of multiple serine to alanine substitutions were consistent with the notion that phosphorylation at individual sites produced roughly additive effects, suggesting that the effect produced by phosphorylation of any one serine was not dependent on the phosphorylation state of other serines. These results are consistent with the notion that, although none of the phosphorylation sites studied here are absolutely necessary for activation of CFTR, individual sites contribute differently to the gating of the channel.

Summary. Transmural fluxes of 3H-mannitol and 22Na or a6CI were measured simultaneously in portions of isolated turtle colon stripped of serosal musculature. The relationships between mannitol flux and the flux of Na or C1 are characteristic of simple diffusion and suggest that transmural mannitol flow is largely confined to a paracellular pathway where Na, C1 and mannitol move much as in free solution. The contribution of"edge damage" to the transmural mannitol flow appears to be minimal. Mucosal hyperosmolarity causes "blisters" in epithelial tight junctions and increases the diffusional permeability to Na and mannitol, suggesting that the rate-limiting barrier in the shunt path is the tight junction. If the total mucosa to serosa flux of Na is corrected for the portion traversing the shunt pathway it is apparent that changes in the short-circuit current are completely accounted for by the mucosa to serosal movement of Na through a cellular path. In addition, the serosa to macosa flux of Na appears to be restricted to the shunt. These observations suggest that there is no appreciable "backflux" of Na through the active, cellular path. In the presence of 10-4 M amiloride the short-circuit current is markedly reduced and the mucosa to serosa Na flux is restricted to the shunt, so that the net Na flux is abolished. The small amiloride-insensitive short-circuit current is consistent with HCO 3 secretion. Mucosa to serosa and serosa to mucosa fluxes of C1 appear to be largely restricted to the paracellular shunt path and there is no evidence for any net flow of C1 under short-circuit conditions. The total tissue conductance can be described as the sum of three components: a shunt conductance which is linearly related to the transmural mannitol flow, an "active" conductance which is linearly related to the short-circuit current and a small residual conductance. The shunt conductance is attributable to the diffusive movements of Na and C1 through the paracellular path. Variations in the active Na transport from tissue to tissue are largely attributable to variations in the apparent conductance of the active Na transport path. The driving force for active Na transport can be described as an apparent emf of approximately 130 mV. These results suggest that transmural mannitol flux provides a quantitative estimate of the ion permeability and electrical conductance of a paracellular shunt path across the isolated turtle colon and thereby facilitates the study of the transport characteristics and electrical properties of cellular paths for transepithelial solute movement.The simplest model which can account for the transport properties of an epithelial cell layer is one which allows for two parallel paths for transmural solute and water flow, one through the cells and another between