Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Research Articles

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.

The molecular evolution of function in the CFTR chloride channel.

The ATP-binding cassette (ABC) transporter superfamily includes many proteins of clinical relevance, with genes expressed in all domains of life. Although most members use the energy of ATP binding and hydrolysis to accomplish the active import or export of various substrates across membranes, the cystic fibrosis transmembrane conductance regulator (CFTR) is the only known animal ABC transporter that functions primarily as an ion channel. Defects in CFTR, which is closely related to ABCC subfamily members that bear function as bona fide transporters, underlie the lethal genetic disease cystic fibrosis. This article seeks to integrate structural, functional, and genomic data to begin to answer the critical question of how the function of CFTR evolved to exhibit regulated channel activity. We highlight several examples wherein preexisting features in ABCC transporters were functionally leveraged as is, or altered by molecular evolution, to ultimately support channel function. This includes features that may underlie (1) construction of an anionic channel pore from an anionic substrate transport pathway, (2) establishment and tuning of phosphoregulation, and (3) optimization of channel function by specialized ligand-channel interactions. We also discuss how divergence and conservation may help elucidate the pharmacology of important CFTR modulators.

Review of CFTR modulators 2020.

Cystic fibrosis transmembrane conductance regulator (CFTR) modulators are small molecules that directly impact the CFTR protein, improving the function of the CFTR chloride and bicarbonate channel. Beginning in 2012 with the Food and Drug Administration approval of the first CFTR modulator, ivacaftor, this class of medications has had largely positive effects on many outcomes in people with cystic fibrosis (PwCF), including lung function, growth, and other clinical parameters. There have been continued exciting developments in the current research on CFTR modulators, expanding beyond original studies. This first part of a three-part cystic fibrosis (CF) year in review 2020 will focus on research on CFTR modulators. In addition to reviewing new clinical insights, we describe work done on novel outcomes, adverse effects, issues related to cost, and next steps for clinical trials. The review focuses on articles from Pediatric Pulmonology published in 2020, but it includes articles from other journals that are of particular interest to clinicians. New developments in CF research continue to be brought forth to the CF community, deepening the understanding of this disease and improving clinical care.

Potential of Intestinal Current Measurement for Personalized Treatment of Patients with Cystic Fibrosis.

Refinement of personalized treatment of cystic fibrosis (CF) with emerging medicines targeting the CF basic defect will likely benefit from biomarkers sensitive to detect improvement of cystic fibrosis transmembrane conductance regulator (CFTR) function in individual patients. Intestinal current measurement (ICM) is a technique that enables quantitative assessment of CFTR chloride channel function in rectal tissues or other intestinal epithelia. ICM was originally developed to study the CF ion transport defect in the intestine and has been established as a sensitive biomarker of CFTR function and diagnostic test for CF. With the emergence of CFTR-directed therapeutics, ICM has become an important tool to estimate the level of rescue of CFTR function achieved by approved CFTR modulators, both at the level of CFTR genotype groups, as well as individual patients with CF. In combination with preclinical patient-derived cell culture models, ICM may aid the development of targeted therapies for patients with rare CFTR mutations. Here, we review the principles of ICM and examine how this CFTR biomarker may be used to support diagnostic testing and enhance personalized medicine for individual patients with common as well as rare CFTR mutations in the new era of medicines targeting the underlying cause of CF.

Bone Cells Differentiation: How CFTR Mutations May Rule the Game of Stem Cells Commitment?

Cystic fibrosis (CF)-related bone disease has emerged as a significant comorbidity of CF and is characterized by decreased bone formation and increased bone resorption. Both osteoblast and osteoclast differentiations are impacted by cystic fibrosis transmembrane conductance regulator (CFTR) mutations. The defect of CFTR chloride channel or the loss of CFTRs ability to interact with other proteins affect several signaling pathways involved in stem cell differentiation and the commitment of these cells toward bone lineages. Specifically, TGF-, nuclear factor-kappa B (NF-B), PI3K/AKT, and MAPK/ERK signaling are disturbed by CFTR mutations, thus perturbing stem cell differentiation. High inflammation in patients changes myeloid lineage secretion, affecting both myeloid and mesenchymal differentiation. In osteoblast, Wnt signaling is impacted, resulting in consequences for both bone formation and resorption. Finally, CFTR could also have a direct role in osteoclasts resorptive function. In this review, we summarize the existing literature on the role of CFTR mutations on the commitment of induced pluripotent stem cells to bone cells.

Identification of Compounds That Promote Readthrough of Premature Termination Codons in the CFTR.

Cystic fibrosis (CF) is caused by a mutation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, which disrupts an ion channel involved in hydration maintenance via anion homeostasis. Nearly 5% of CF patients possess one or more copies of the G542X allele, which results in a stop codon at residue 542, preventing full-length CFTR protein synthesis. Identifying small-molecule modulators of mutant CFTR biosynthesis that affect the readthrough of this and other premature termination codons to synthesize a fully functional CFTR protein represents a novel target area of drug discovery. We describe the implementation and integration for large-scale screening of a homogeneous, 1536-well functional G542X-CFTR readthrough assay. The assay uses HEK 293 cells engineered to overexpress the G542X-CFTR mutant, whose functional activity is monitored with a membrane potential dye. Cells are co-incubated with a CFTR amplifier and CFTR corrector to maximize mRNA levels and trafficking of CFTR to the cell surface. Compounds that allow translational readthrough and synthesis of functional CFTR chloride channels are reflected by changes in membrane potential in response to cAMP stimulation with forskolin and CFTR channel potentiation with genistein. Assay statistics yielded Z′ values of 0.69 ± 0.06. As further evidence of its suitability for high-throughput screening, we completed automated screening of approximately 666,000 compounds, identifying 7761 initial hits. Following secondary and tertiary assays, we identified 188 confirmed hit compounds with low and submicromolar potencies. Thus, this approach takes advantage of a phenotypic screen with high-throughput scalability to identify new small-molecule G542X-CFTR readthrough modulators.

Ionocytes and CFTR Chloride Channel Expression in Normal and Cystic Fibrosis Nasal and Bronchial Epithelial Cells.

The airway epithelium contains ionocytes, a rare cell type with high expression of Forkhead Box I1 (FOXI1) transcription factor and Cystic Fibrosis Transmembrane conductance Regulator (CFTR), a chloride channel that is defective in cystic fibrosis (CF). Our aim was to verify if ionocyte development is altered in CF and to investigate the relationship between ionocytes and CFTR-dependent chloride secretion. We collected nasal cells by brushing to determine ionocyte abundance. Nasal and bronchial cells were also expanded in vitro and reprogrammed to differentiated epithelia for morphological and functional studies. We found a relatively high (~3%) ionocyte abundance in ex vivo nasal samples, with no difference between CF and control individuals. In bronchi, ionocytes instead appeared very rarely as previously reported, thus suggesting a possible proximal–distal gradient in human airways. The difference between nasal and bronchial epithelial cells was maintained in culture, which suggests an epigenetic control of ionocyte development. In the differentiation phase of the culture procedure, we used two media that resulted in a different pattern of CFTR expression: confined to ionocytes or more broadly expressed. CFTR function was similar in both conditions, thus indicating that chloride secretion equally occurs irrespective of CFTR expression pattern.

The Role of Specialized Pro-Resolving Mediators in Cystic Fibrosis Airways Disease.

Cystic Fibrosis (CF) is a recessive genetic disease due to mutations of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene encoding the CFTR chloride channel. The ion transport abnormalities related to CFTR mutation generate a dehydrated airway surface liquid (ASL) layer, which is responsible for an altered mucociliary clearance, favors infections and persistent inflammation that lead to progressive lung destruction and respiratory failure. The inflammatory response is normally followed by an active resolution phase to return to tissue homeostasis, which involves specialized pro-resolving mediators (SPMs). SPMs promote resolution of inflammation, clearance of microbes, tissue regeneration and reduce pain, but do not evoke unwanted immunosuppression. The airways of CF patients showed a decreased production of SPMs even in the absence of pathogens. SPMs levels in the airway correlated with CF patients’ lung function. The prognosis for CF has greatly improved but there remains a critical need for more effective treatments that prevent excessive inflammation, lung damage, and declining pulmonary function for all CF patients. This review aims to highlight the recent understanding of CF airway inflammation and the possible impact of SPMs on functions that are altered in CF airways.

Disrupted local innervation results in less VIP expression in CF mice tissues

Vasoactive Intestinal Peptide (VIP) is the major physiological agonist of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) chloride channel activity. VIP functions as a neuromodulator and neurotransmitter secreted by neurons innervating all exocrine glands. VIP is also a potent vasodilator and bronchodilator that regulates exocrine gland secretions, contributing to local innate defense by stimulating the movement of water and chloride transport across intestinal and tracheobronchial epithelia. Previous human studies have shown that the rich intrinsic neuronal networks for VIP secretion around exocrine glands could be lost in tissues from patients with cystic fibrosis. Our research has since confirmed, in vitro and in vivo, the need for chronic VIP exposure to maintain functional CFTR chloride channels at the cell surface of airways and intestinal epithelium, as well as normal exocrine tissues morphology [1]. The goal of the present study was to examine changes in VIP in the lung, duodenum and sweat glands of 8- and 17-weeks old F508del/F508del mice and to investigate VIPergic innervation in the small intestine of CF mice, before important signs of the disease development. Our data show that a low amount of VIP is found in CF tissues prior to tissue damage. Moreover, we found a specific reduction in VIPergic and cholinergic innervation of the small intestine. The general innervation of the primary and secondary myenteric plexus was lost in CF tissues, with the presence of enlarged ganglionic cells in the tertiary layer. We propose that low amount of VIP in CF tissues is due to a reduction in VIPergic and cholinergic innervation and represents an early defect that constitutes an aggravating factor for CF disease progression.

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.

Nonsense-mediated RNA Decay Pathway Inhibition Restores Expression and Function of W1282X CFTR.

The recessive genetic disease cystic fibrosis (CF) is caused by loss-of-function mutations in the CFTR (CF transmembrane conductance regulator) gene. Approximately 10% of patients with CF have at least one allele with a nonsense mutation in CFTR. Nonsense mutations generate premature termination codons that can subject mRNA transcripts to rapid degradation through the nonsense-mediated mRNA decay (NMD) pathway. Currently, there are no approved therapies that specifically target nonsense mutations in CFTR. Here, we identified antisense oligonucleotides (ASOs) that target the NMD factor SMG1 to inhibit the NMD pathway, and determined their effects on the W1282X CFTR mutation. First, we developed and validated two in vitro models of the W1282X CFTR mutation. Next, we treated these cells with antisense oligonucleotides to inhibit NMD and measured the effects of these treatments on W1282X expression and function. SMG1-ASO-mediated NMD inhibition upregulated the RNA, protein, and surface-localized protein expression of the truncated W1282X gene product. Additionally, these ASOs increased the CFTR chloride channel function in cells homozygous for the W1282X mutation. Our approach suggests a new therapeutic strategy for patients harboring nonsense mutations and may be beneficial as a single agent in patients with CF and the W1282X mutation.

Azaspiracids Increase Mitochondrial Dehydrogenases Activity in Hepatocytes: Involvement of Potassium and Chloride Ions

Background: Azaspiracids (AZAs) are marine toxins that are produced by Azadinium and Amphidoma dinoflagellates that can contaminate edible shellfish inducing a foodborne poisoning in humans, which is characterized by gastrointestinal symptoms. Among these, AZA1, -2, and -3 are regulated in the European Union, being the most important in terms of occurrence and toxicity. In vivo studies in mice showed that, in addition to gastrointestinal effects, AZA1 induces liver alterations that are visible as a swollen organ, with the presence of hepatocellular fat droplets and vacuoles. Hence, an in vitro study was carried out to investigate the effects of AZA1, -2, and -3 on liver cells, using human non-tumor IHH hepatocytes. Results: The exposure of IHH cells to AZA1, -2, or -3 (5 × 10−12–1 × 10−7 M) for 24 h did not affect the cell viability and proliferation (Sulforhodamine B assay and 3H-Thymidine incorporation assay), but they induced a significant concentration-dependent increase of mitochondrial dehydrogenases activity (MTT reduction assay). This effect depends on the activity of mitochondrial electron transport chain complex I and II, being counteracted by rotenone and tenoyl trifluoroacetone, respectively. Furthermore, AZAs-increased mitochondrial dehydrogenase activity was almost totally suppressed in the K+-, Cl−-, and Na+-free media and sensitive to the specific inhibitors of KATP and hERG potassium channels, Na+/K+, ATPase, and cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels. Conclusions: These results suggest that AZA mitochondrial effects in hepatocytes derive from an imbalance of intracellular levels of K+ and, in particular, Cl− ions, as demonstrated by the selective reduction of toxin effects by CFTR chloride channel inhibition.

A Study on the Effect of N453 from Fungi on Inhibiting CFTR Chloride Channels in Intestinal Epithelial Cells

Secretory diarrhea remains a global health problem, especially in developing countries. Cyclic AMP‐stimulated intestinal Cl− secretion plays a crucial role in pathogenesis of diarrheas including cholera and traveler's diarrhea. Inhibition of cAMP‐activated Cl− channel is considered as drug target for secretory diarrhea. The main objective of this study was to identify a novel inhibitor of the cystic fibrosis transmembrane conductance regulator (CFTR), cAMP‐activated Cl− channel, from a screen of fungal metabolites. A fungal derivative compound N453 was identified as an active compound that could inhibit cAMP‐activated Cl− secretion in intact T84 cells with a half maximal inhibitory concentration (IC50) of ~ 0.78 μM in Ussing chamber experiments. Importantly, MTT assays revealed that N453 (up to100 μM) did not affect cell viability, suggesting that inhibition of cAMP‐activated Cl− current was not due to cytotoxic effect. In apical Cl− current analysis, N453 suppressed CFTR‐mediated apical Cl− current in a concentration‐dependent manner with IC50 of ~ 2 μM. On the other hand, the Na+‐K+ ATPase activity was not affected by N453. Of interest, N453 concentration‐dependently inhibited basolateral cAMP‐activated K+ channels with an IC50 of ~ 23 μM. In conclusion, this study demonstrated that N453 inhibits Cl− secretion in intestinal epithelial cells, at least in part, by inhibiting CFTR‐mediated apical Cl− current and basolateral cAMP‐activated K+ channels. N453 may be useful as potential therapy of secretory diarrheas.Support or Funding InformationThis work was supported by the NSTDA Chair Professor grant (the Fourth Grant) of the Crown Property Bureau Foundation, Faculty of Science Mahidol University, Faculty of Graduate Studies Mahidol University and Tonkla Ramathibodi.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

Resveratrol dimer trans-ε-viniferin prevents rotaviral diarrhea in mice by inhibition of the intestinal calcium-activated chloride channel

We previously identified, by a natural-product screen, resveratrol oligomers as inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. Here, we report the resveratrol dimer trans-ε-viniferin (TV) and tetramer r-2-viniferin (RV) as inhibitors of the intestinal calcium-activated chloride channel (CaCC) and demonstrate their antisecretory efficacy in a neonatal mouse model of rotaviral diarrhea. Short-circuit measurements show inhibition of CaCC current in the human colonic cell line HT-29 by TV and RV with IC50∼1 and 20μM, respectively. TV primarily inhibited the physiologically relevant, long-term CaCC current following agonist stimulation, without effect on cytoplasmic Ca2+ signaling. TV and RV inhibited short-circuit current in mouse colon as well. In a neonatal mouse model of rotaviral secretory diarrhea produced by oral inoculation with rotavirus, 2μg TV or 11μg RV inhibited secretory diarrhea by >50%, without effect on the rotaviral infection. Our results support the antisecretory efficacy of non-toxic, natural-product resveratrol oligomers for diarrheas produced by CaCC activation. Because these compounds also inhibit the CFTR chloride channel, they may be useful for antisecretory therapy of a wide range of diarrheas.

Molecular dynamics of the cryo-EM CFTR structure

Cystic fibrosis (CF), a lethal monogenic disease, is caused by mutant variants of the CF transmembrane conductance regulator (CFTR). Recent advances in single molecule cryo-EM methods enabled structural determination of full-length human and zebrafish CFTR, achieving an important milestone for CF drug development. To relate these structures to the gating cycle, we examined its dynamic features using molecular dynamics simulations. Our results show that the nucleotide binding domains (NBDs) in this bottom-open apo conformation exhibit motions related to dimerization and the bottom-closed apo CFTR model indicates opening of NBDs in contrast to transporters. These observations help in understanding the properties of CFTR chloride channel distinct from transporters and in proper interpretation of available structural information on this ABC protein.

Bacterial Sphingomyelinase is a State-Dependent Inhibitor of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR)

Sphingomyelinase C (SMase) inhibits CFTR chloride channel activity in multiple cell systems, an effect that could exacerbate disease in CF and COPD patients. The mechanism by which sphingomyelin catalysis inhibits CFTR is not known but evidence suggests that it occurs independently of CFTR’s regulatory “R” domain. In this study we utilized the Xenopus oocyte expression system to shed light on how CFTR channel activity is reduced by SMase. We found that the pathway leading to inhibition is not membrane delimited and that inhibited CFTR channels remain at the cell membrane, indicative of a novel silencing mechanism. Consistent with an effect on CFTR gating behavior, we found that altering gating kinetics influenced the sensitivity to inhibition by SMase. Specifically, increasing channel activity by introducing the mutation K1250A or pretreating with the CFTR potentiator VX-770 (Ivacaftor) imparted resistance to inhibition. In primary bronchial epithelial cells, we found that basolateral, but not apical, application of SMase leads to a redistribution of sphingomyelin and a reduction in forskolin- and VX-770-stimulated currents. Taken together, these data suggest that SMase inhibits CFTR channel function by locking channels into a closed state and that endogenous CFTR in HBEs is affected by SMase activity.

Mashiningan Improves Opioid-Induced Constipation in Rats by Activating Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel.

Opioid receptor stimulants are analgesics used in patients with and without cancer; however, they often cause constipation, resulting in poor adherence and deterioration of the quality of life. Hence, suitable treatments for constipation are required. In this study, we investigated the pharmacological mechanisms of action of mashiningan (MNG), a Kampo medicine used to treat constipation, and evaluated the effect of MNG on opioid-induced constipation in rats. MNG (100 or 300 mg/kg) was orally administered to normal or codeine phosphate (CPH)-induced constipation in rats, and its effect was evaluated on the basis of fecal counts, characteristics, and weight. Small intestinal fluid secretion was measured after treatment with MNG alone or coadministration with a cystic fibrosis transmembrane conductance regulator (CFTR)-specific inhibitor (CFTRinh-172). The effects of MNG on the CFTR and type-2 chloride channel were determined using patch-clamp or short-circuit current experiments, respectively. MNG increased the fecal weight and proportion of soft feces in normal rats. CPH-induced constipation in rats decreased fecal counts and weight, whereas MNG prevented these effects and increased the proportion of soft feces. MNG increased the electronic chloride current, and this effect was inhibited by the CFTRinh-172 in the CFTR assay. Furthermore, MNG increased small intestinal fluid secretion, and this effect was abolished by coadministration with the CFTRinh-172. MNG improved opioid-induced constipation in rats, and this improvement may have been mediated by increasing intestinal fluid secretion via CFTR chloride channel activation. Therefore, MNG is expected as a medicine of the treatment of constipation in patients taking opioids.

Synergy of cAMP and calcium signaling pathways in CFTR regulation

Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, leading to defective apical chloride transport. Patients also experience overactivation of inflammatory processes, including increased calcium signaling. Many investigations have described indirect effects of calcium signaling on CFTR or other calcium-activated chloride channels; here, we investigate the direct response of CFTR to calmodulin-mediated calcium signaling. We characterize an interaction between the regulatory region of CFTR and calmodulin, the major calcium signaling molecule, and report protein kinase A (PKA)-independent CFTR activation by calmodulin. We describe the competition between calmodulin binding and PKA phosphorylation and the differential effects of this competition for wild-type CFTR and the major F508del mutant, hinting at potential therapeutic strategies. Evidence of CFTR binding to isolated calmodulin domains/lobes suggests a mechanism for the role of CFTR as a molecular hub. Together, these data provide insights into how loss of active CFTR at the membrane can have additional consequences besides impaired chloride transport.

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.