Trivalent anions as probes of the CFTR channel pore.

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel uses positively charged amino-acid side-chains to form binding sites for permeating anions. These binding sites have been investigated experimentally using a number of anionic probes. Mutations that alter the distribution of positive and negative charges within the pore have differential effects on the binding of monovalent versus divalent anions. This study uses patch clamp recording from wild-type and pore-mutant forms of CFTR to investigate small trivalent anions (Co(NO2)63-, Co(CN)3- and IrCl63-) as potential probes of anion binding sites. These anions caused weak block of Cl- permeation in wild-type CFTR (Kd ≥ 700 µM) when applied to the intracellular side of the membrane. Mutations that increase the density of positive charge within the pore (E92Q, I344K, S1141K) increased the binding affinity of these anions 80-280-fold, and also greatly increased the voltage-dependence of block, consistent with fixed charges in the pore affecting monovalent : multivalent anion selectivity. However, high-affinity pore block by Co(NO2)63-apparently did not alter channel gating, a hallmark of high-affinity binding of divalent Pt(NO2)42- ions within the pore. This work increases the arsenal of probes available to investigate anion binding sites within Cl- channel pores.

Role of Hydrophobic Amino-Acid Side-Chains in the Narrow Selectivity Filter of the CFTR Chloride Channel Pore in Conductance and Selectivity.

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. Structural analysis of CFTR has identified a narrow, hydrophobic region close to the extracellular end of the open channel pore that may function as a selectivity filter. The present study combines comprehensive mutagenesis of hydrophobic amino-acid side-chains within the selectivity filter with functional evaluation of channel Cl- conductance and anion selectivity. Among these hydrophobic amino-acids, one (F337) appears to play a dominant role in determining both conductance and selectivity. Anion selectivity appears to depend on both side-chain size and hydrophobicity at this position. In contrast, conductance is disrupted by all F337 mutations, suggesting that unique interactions between permeating Cl- ions and the native phenylalanine side-chain are important for conductance. Surprisingly, a positively charged lysine side-chain can be substituted for several hydrophobic residues within the selectivity filter (including F337) with only minor changes in pore function, arguing against a crucial role for overall hydrophobicity. These results suggest that localized interactions between permeating anions and amino-acid side-chains within the selectivity filter may be more important in determining pore functional properties than are global features such as overall hydrophobicity.

Molecular dynamics study of Cl- permeation through cystic fibrosis transmembrane conductance regulator (CFTR).

The recent elucidation of atomistic structures of Cl- channel CFTR provides opportunities for understanding the molecular basis of cystic fibrosis. Despite having been activated through phosphorylation and provided with ATP ligands, several near-atomistic cryo-EM structures of CFTR are in a closed state, as inferred from the lack of a continuous passage through a hydrophobic bottleneck region located in the extracellular portion of the pore. Here, we present repeated, microsecond-long molecular dynamics simulations of human CFTR solvated in a lipid bilayer and aqueous NaCl. At equilibrium, Cl- ions enter the channel through a lateral intracellular portal and bind to two distinct cationic sites inside the channel pore but do not traverse the narrow, de-wetted bottleneck. Simulations conducted in the presence of a strong hyperpolarizing electric field led to spontaneous Cl- translocation events through the bottleneck region of the channel, suggesting that the protein relaxed to a functionally open state. Conformational changes of small magnitude involving transmembrane helices 1 and 6 preceded ion permeation through diverging exit routes at the extracellular end of the pore. The pore bottleneck undergoes wetting prior to Cl- translocation, suggesting that it acts as a hydrophobic gate. Although permeating Cl- ions remain mostly hydrated, partial dehydration occurs at the binding sites and in the bottleneck. The observed Cl- pathway is largely consistent with the loci of mutations that alter channel conductance, anion binding, and ion selectivity, supporting the model of the open state of CFTR obtained in the present study.

Functionally additive fixed positive and negative charges in the CFTR channel pore control anion binding and conductance.

Ion channels use charged amino-acid residues to attract oppositely charged permeant ions into the channel pore. In the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel, a number of arginine and lysine residues have been shown to be important for Cl- permeation. Among these, two in close proximity in the pore-Lys95 and Arg134-are indispensable for anion binding and high Cl- conductance, suggesting that high positive charge density is required for pore function. Here we used mutagenesis and functional characterization to show that a nearby pore-lining negatively charged residue (Glu92) plays a functionally additive role with these two positive charges. While neutralization of this negative charge had little effect on anion binding or Cl- conductance, such neutralization was able to reverse the detrimental effects of removing the positive charge at either Lys95 or Arg134, as well as the similar effects of introducing a negative charge at a neighboring residue (Ser1141). Furthermore, neutralization of Glu92 greatly increased the susceptibility of the channel to blockage by divalent S2O32- anions, mimicking the effect of introducing additional positive charge in this region; this effect was reversed by concurrent neutralization of either Lys95 or Arg134. Across a panel of mutant channels that introduced or removed fixed charges at these four positions, we found that many pore properties are dependent on the overall charge or charge density. We propose that the CFTR pore uses a combination of positively and negatively charged residues to optimize the anion binding and Cl- conductance properties of the channel.

Two positively charged amino acid side-chains in the inner vestibule of the CFTR channel pore play analogous roles in controlling anion binding and anion conductance.

Positively charged amino acid side-chains play important roles in anion binding and permeation through the CFTR chloride channel. One pore-lining lysine residue in particular (K95) has been shown to be indispensable for anion binding, conductance, and selectivity. Here, we use functional investigation of CFTR to show that a nearby arginine (R134) plays a functionally analogous role. Removal of this positive charge (in the R134Q mutant) drastically reduces single-channel conductance, weakens binding of both permeant and blocking anions, and abolishes the normal anion conductance selectivity pattern. Each of these functional effects was reversed by a second-site mutation (S1141K) that introduces an ectopic positive charge to a nearby pore-lining residue. Substituted cysteine accessibility experiments confirm that R134-but not nearby residues in the same transmembrane helix-is accessible within the pore lumen. These results suggest that K95 and R134, which are very close together within the inner vestibule of the pore, play analogous, important roles, and that both are required for the normal anion binding and anion conductance properties of the pore. Nevertheless, that fact that both positive charges can be "transplanted" to other sites in the inner vestibule with little effect on channel permeation properties indicates that it is the overall number of charges-rather than their exact locations-that controls pore function.

Monovalent: Divalent Anion Selectivity in the CFTR Channel Pore.

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel shows only weak selectivity between different small monovalent anions, however, little is known about its ability to discriminate between monovalent and divalent anions. The present study uses patch clamp recording to investigate the interaction between the small divalent anions S2O32- and SO42- and wild-type and pore-mutant forms of human CFTR. Binding of these anions to wild-type CFTR appears weak; at 10 mM, intracellular S2O32- and SO42- blocked <20 and <5% of macroscopic Cl- current respectively, while these same concentrations had no discernible blocking effect when present in the extracellular solution. However, introduction of additional positive charge into the inner vestibule of the pore (in I344K and S1141K mutant channels) drastically strengthened block by intracellular (but not extracellular) S2O32- and SO42-. Block of these mutant channels was highly voltage-dependent; at very negative membrane potentials, apparent binding affinities were ~100 µM for S2O32- and <1 mM for SO42-. Permeability of S2O32- and SO42- was too small to be quantified in wild-type CFTR, but was <1% of Cl- permeability. Mutants that strengthened divalent binding (I344K, S1141K), as well as the selectivity-altering mutant F337A, also showed immeasurably low S2O32- and SO42- permeabilities. Overall CFTR selects well for monovalent over divalent anions, both in terms of binding and permeability. The number or density of fixed positive charges in the pore appears well optimized to disfavour binding of divalent anions, which may be an important facet of the monovalent Cl- permeation mechanism.

On the relationship between anion binding and chloride conductance in the CFTR anion channel.

Mutations at many sites within the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel pore region result in changes in chloride conductance. Although chloride binding in the pore - as well as interactions between concurrently bound chloride ions - are thought to be important facets of the chloride permeation mechanism, little is known about the relationship between anion binding and chloride conductance. The present work presents a comprehensive investigation of a number of anion binding properties in different pore mutants with differential effects on chloride conductance. When multiple pore mutants are compared, conductance appears best correlated with the ability of anions to bind to the pore when it is already occupied by chloride ions. In contrast, conductance was not correlated with biophysical measures of anion:anion interactions inside the pore. Although these findings suggest anion binding is required for high conductance, mutations that strengthened anion binding had very little effect on conductance, especially at high chloride concentrations, suggesting that the wild-type CFTR pore is already close to saturated with chloride ions. These results are used to support a revised model of chloride permeation in CFTR in which the overall chloride occupancy of multiple loosely-defined chloride binding sites results in high chloride conductance through the pore.

Electrostatic Tuning of Anion Attraction from the Cytoplasm to the Pore of the CFTR Chloride Channel.

Anions enter from the cytoplasm into the channel pore of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel not via a central pathway but via a single lateral portal or fenestration. High Cl- conductance is dependent on electrostatic attraction of cytoplasmic Cl- ions by four positively charged amino acid side-chains located within this portal. Here we use a mutagenic approach to investigate the functional effects of transplanting or supplementing these positive charges at nearby portal-lining sites. Using patch clamp recording, we find that the functionally important positive charges at K190 and R303 can be transplanted to four nearby sites (N186, L197, W356, and A367) with little loss of Cl- conductance. Introduction of additional positive charge at these sites had almost no effect on Cl- conductance, but did increase the sensitivity to channel block by intracellular suramin and Pt(NO2)42- anions. We suggest that it is the number of positive charges within the portal, rather than their exact location, that is the most important factor influencing Cl- conductance. The portal appears well optimized in terms of charge distribution to maximize Cl- conductance.

Contribution of the eighth transmembrane segment to the function of the CFTR chloride channel pore.

Our molecular understanding of the cystic fibrosis transmembrane conductance regulator (CFTR)-the chloride channel that is mutated in cystic fibrosis-has been greatly enhanced by a number of recent atomic-level structures of the protein in different conformations. One surprising aspect of these structures was the finding that the eighth of CFTR's 12 membrane-spanning segments (TM8) appeared close to the channel pore. Although functional evidence supports a role for other TMs in forming the pore, such a role for TM8 has not previously been reported. Here, we use patch-clamp recording to investigate the functional role of TM8. Using substituted cysteine accessibility mutagenesis, we find that three amino acid side-chains in TM8 (Y913, Y914, and Y917) are exposed to the extracellular, but not the intracellular, solution. Cysteine cross-linking experiments suggest that Y914 and Y917 are in close proximity to L102 (TM1) and F337 (TM6), respectively, suggesting that TM8 contributes to the narrow selectivity filter region of the pore. Different amino acid substitutions suggest that Y914, and to a lesser extent Y917, play important roles in controlling anion flux through the open channel. Furthermore, substitutions that reduce side-chain volume at Y917 severely affect channel gating, resulting in a channel with an extremely unstable open state. Our results suggest that pore-lining TM8 is among the most important TMs controlling the permeation phenotype of the CFTR channel, and also that movement of TM8 may be critically involved in channel gating.

Cystic fibrosis transmembrane conductance regulator (CFTR): Making an ion channel out of an active transporter structure.

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is a member of the ATP-binding cassette (ABC) family of membrane transport proteins, most members of which function as ATP-dependent pumps. CFTR is unique among human ABC proteins in functioning not as a pump, but as an ion channel. Recent structural data has indicated that CFTR shares broadly similar overall architecture and ATP-dependent conformational changes as other ABC proteins. Functional investigations suggest that CFTR has a unique open portal connecting the cytoplasm to the transmembrane channel pore, that allows for a continuous pathway for Cl- ions to cross the membrane in one conformation. This lateral portal may be what allows CFTR to function as an ion channel rather than as a pump, suggesting a plausible mechanism by which channel function may have evolved in CFTR.

Functional organization of cytoplasmic portals controlling access to the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel pore.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel that apparently has evolved from an ancestral active transporter. Key to the CFTR's switch from pump to channel function may have been the appearance of one or more "lateral portals." Such portals connect the cytoplasm to the transmembrane channel pore, allowing a continuous pathway for the electrodiffusional movement of Cl- ions. However, these portals remain the least well-characterized part of the Cl- transport pathway; even the number of functional portals is uncertain, and if multiple portals do exist, their relative functional contributions are unknown. Here, we used patch-clamp recording to identify the contributions of positively charged amino acid side chains located in CFTR's cytoplasmic transmembrane extensions to portal function. Mutagenesis-mediated neutralization of several charged side chains reduced single-channel Cl- conductance. However, these same mutations differentially affected channel blockade by cytoplasmic suramin and Pt(NO2)42- anions. We considered and tested several models by which the contribution of these positively charged side chains to one or more independent or non-independent portals to the pore could affect Cl- conductance and interactions with blockers. Overall, our results suggest the existence of a single portal that is lined by several positively charged side chains that interact electrostatically with both Cl- and blocking anions. We further propose that mutations at other sites indirectly alter the function of this single portal. Comparison of our functional results with recent structural information on CFTR completes our picture of the overall molecular architecture of the Cl- permeation pathway.

Conformational change of the extracellular parts of the CFTR protein during channel gating.

Cystic fibrosis can be treated by potentiators, drugs that interact directly with the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel to increase its open probability. These substances likely target key conformational changes occurring during channel opening and closing, however, the molecular bases of these conformational changes, and their susceptibility to manipulation are poorly understood. We have used patch clamp recording to identify changes in the three-dimensional organization of the extracellularly accessible parts of the CFTR protein during channel opening and closing. State-dependent formation of both disulfide bonds and Cd2+ bridges occurred for pairs of cysteine side-chains introduced into the extreme extracellular ends of transmembrane helices (TMs) 1, 6, and 12. Between each of these three TMs, we found that both disulfide bonds and metal bridges formed preferentially or exclusively in the closed state and that these inter-TM cross-links stabilized the closed state. These results indicate that the extracellular ends of these TMs are close together when the channel is closed and that they separate from each other when the channel opens. These findings identify for the first time key conformational changes in the extracellular parts of the CFTR protein that can potentially be manipulated to control channel activity.

Contribution of a leucine residue in the first transmembrane segment to the selectivity filter region in the CFTR chloride channel.

The anion selectivity and conductance of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel are determined predominantly by interactions between permeant anions and the narrow region of the channel pore. This narrow region has therefore been described as functioning as the "selectivity filter" of the channel. Multiple pore-lining transmembrane segments (TMs) have previously been shown to contribute to the selectivity filter region. However, little is known about the three-dimensional organization of this region, or how multiple TMs combine to determine its functional properties. In the present study we have used patch clamp recording to identify changes in channel function associated with the formation of disulfide cross-links between cysteine residues introduced into different TMs within the selectivity filter. Cysteine introduced at position L102 in TM1 was able to form disulfide bonds with F337C and T338C in TM6, two positions that are known to play key roles in determining anion permeation properties. Consistent with this proximal arrangement of L102, F337 and T338, different mutations at L102 altered anion selectivity and conductance properties in a way that suggests that this residue plays an important role in determining selectivity filter function, albeit a much lesser role than that of F337. These results suggest an asymmetric three-dimensional arrangement of the key selectivity filter region of the pore, as well as having important implications regarding the molecular mechanism of anion permeation.

Structural Changes Fundamental to Gating of the Cystic Fibrosis Transmembrane Conductance Regulator Anion Channel Pore.

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial cell anion channel. Potentiator drugs used in the treatment of cystic fibrosis act on the channel to increase overall channel function, by increasing the stability of its open state and/or decreasing the stability of its closed state. The structure of the channel in either the open state or the closed state is not currently known. However, changes in the conformation of the protein as it transitions between these two states have been studied using functional investigation and molecular modeling techniques. This review summarizes our current understanding of the architecture of the transmembrane channel pore that controls the movement of chloride and other small anions, both in the open state and in the closed state. Evidence for different kinds of changes in the conformation of the pore as it transitions between open and closed states is described, as well as the mechanisms by which these conformational changes might be controlled to regulate normal channel gating. The ways that key conformational changes might be targeted by small compounds to influence overall CFTR activity are also discussed. Understanding the changes in pore structure that might be manipulated by such small compounds is key to the development of novel therapeutic strategies for the treatment of cystic fibrosis.

Architecture and functional properties of the CFTR channel pore.

The main function of the cystic fibrosis transmembrane conductance regulator (CFTR) is as an ion channel for the movement of small anions across epithelial cell membranes. As an ion channel, CFTR must form a continuous pathway across the cell membrane-referred to as the channel pore-for the rapid electrodiffusional movement of ions. This review summarizes our current understanding of the architecture of the channel pore, as defined by electrophysiological analysis and molecular modeling studies. This includes consideration of the characteristic functional properties of the pore, definition of the overall shape of the entire extent of the pore, and discussion of how the molecular structure of distinct regions of the pore might control different facets of pore function. Comparisons are drawn with closely related proteins that are not ion channels, and also with structurally unrelated proteins with anion channel function. A simple model of pore function is also described.

Anion conductance selectivity mechanism of the CFTR chloride channel.

All ion channels are able to discriminate between substrate ions to some extent, a process that involves specific interactions between permeant anions and the so-called selectivity filter within the channel pore. In the cystic fibrosis transmembrane conductance regulator (CFTR) anion-selective channel, both anion relative permeability and anion relative conductance are dependent on anion free energy of hydration--anions that are relatively easily dehydrated tend to show both high permeability and low conductance. In the present work, patch clamp recording was used to investigate the relative conductance of different anions in CFTR, and the effect of mutations within the channel pore. In constitutively-active E1371Q-CFTR channels, the anion conductance sequence was Cl(-) > NO3(-) > Br(-) > formate > SCN(-) > I(-). A mutation that disrupts anion binding in the inner vestibule of the pore (K95Q) disrupted anion conductance selectivity, such that anions with different permeabilities showed almost indistinguishable conductances. Conversely, a mutation at the putative narrowest pore region that is known to disrupt anion permeability selectivity (F337A) had minimal effects on anion relative conductance. Ion competition experiments confirmed that relatively tight binding of permeant anions resulted in relatively low conductance. These results suggest that the relative affinity of ion binding in the inner vestibule of the pore controls the relative conductance of different permeant anions in CFTR, and that the pore has two physically distinct anion selectivity filters that act in series to control anion conductance selectivity and anion permeability selectivity respectively.

Cytoplasmic pathway followed by chloride ions to enter the CFTR channel pore.

Most ATP-binding cassette (ABC) proteins function as ATP-dependent membrane pumps. One exception is the cystic fibrosis transmembrane conductance regulator (CFTR), an ABC protein that functions as a Cl(-) ion channel. As such, the CFTR protein must form a continuous pathway for the movement of Cl(-) ions from the cytoplasm to the extracellular solution when in its open channel state. Extensive functional investigations have characterized most parts of this Cl(-) permeation pathway. However, one region remains unexplored-the pathway connecting the cytoplasm to the membrane-spanning pore. We used patch clamp recording and extensive substituted cysteine accessibility mutagenesis to identify amino acid side-chains in cytoplasmic regions of CFTR that lie close to the pathway taken by Cl(-) ions as they pass from the cytoplasm through this pathway. Our results suggest that Cl(-) ions enter the permeation pathway via a single lateral tunnel formed by the cytoplasmic parts of the protein, and then follow a fairly direct central pathway towards the membrane-spanning parts of the protein. However, this pathway is not lined continuously by any particular part of the protein; instead, the contributions of different cytoplasmic regions of the protein appear to change as the permeation pathway approaches the membrane, which appears to reflect the ways in which different cytoplasmic regions of the protein are oriented towards its central axis. Our results allow us to define for the first time the complete Cl(-) permeation pathway in CFTR, from the cytoplasm to the extracellular solution.

Metal bridges to probe membrane ion channel structure and function.

Ion channels are integral membrane proteins that undergo important conformational changes as they open and close to control transmembrane flux of different ions. The molecular underpinnings of these dynamic conformational rearrangements are difficult to ascertain using current structural methods. Several functional approaches have been used to understand two- and three-dimensional dynamic structures of ion channels, based on the reactivity of the cysteine side-chain. Two-dimensional structural rearrangements, such as changes in the accessibility of different parts of the channel protein to the bulk solution on either side of the membrane, are used to define movements within the permeation pathway, such as those that open and close ion channel gates. Three-dimensional rearrangements ­ in which two different parts of the channel protein change their proximity during conformational changes ­ are probed by cross-linking or bridging together two cysteine side-chains. Particularly useful in this regard are so-called metal bridges formed when two or more cysteine side-chains form a high-affinity binding site for metal ions such as Cd2+ or Zn2+. This review describes the use of these different techniques for the study of ion channel dynamic structure and function, including a comprehensive review of the different kinds of conformational rearrangements that have been studied in different channel types via the identification of intra-molecular metal bridges. Factors that influence the affinities and conformational sensitivities of these metal bridges, as well as the kinds of structural inferences that can be drawn from these studies, are also discussed.

Functional Architecture of the Cytoplasmic Entrance to the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Pore.

As an ion channel, the cystic fibrosis transmembrane conductance regulator must form a continuous pathway for the movement of Cl(-) and other anions between the cytoplasm and the extracellular solution. Both the structure and the function of the membrane-spanning part of this pathway are well defined. In contrast, the structure of the pathway that connects the cytoplasm to the membrane-spanning regions is unknown, and functional roles for different parts of the protein forming this pathway have not been described. We used patch clamp recording and substituted cysteine accessibility mutagenesis to identify positively charged amino acid side chains that attract cytoplasmic Cl(-) ions to the inner mouth of the pore. Our results indicate that the side chains of Lys-190, Arg-248, Arg-303, Lys-370, Lys-1041, and Arg-1048, located in different intracellular loops of the protein, play important roles in the electrostatic attraction of Cl(-) ions. Mutation and covalent modification of these residues have charge-dependent effects on the rate of Cl(-) permeation, demonstrating their functional role in maximization of Cl(-) flux. Other nearby positively charged side chains were not involved in electrostatic interactions with Cl(-). The location of these Cl(-)-attractive residues suggests that cytoplasmic Cl(-) ions enter the pore via a lateral portal located between the cytoplasmic extensions to the fourth and sixth transmembrane helices; a secondary, functionally less relevant portal might exist between the extensions to the 10th and 12th transmembrane helices. These results define the cytoplasmic mouth of the pore and show how it attracts Cl(-) ions from the cytoplasm.