Claudin family tight junction proteins form charge- and size-selective paracellular channels that regulate epithelial barrier function. In the gastrointestinal tract, barrier heterogeneity is attributed to differential claudin expression. Here, we show that claudin-23 (CLDN23) is enriched in luminal intestinal epithelial cells where it strengthens the epithelial barrier. Complementary approaches reveal that CLDN23 regulates paracellular ion and macromolecule permeability by associating with CLDN3 and CLDN4 and regulating their distribution in tight junctions. Computational modeling suggests that CLDN23 forms heteromeric and heterotypic complexes with CLDN3 and CLDN4 that have unique pore architecture and overall net charge. These computational simulation analyses further suggest that pore properties are interaction-dependent, since differently organized complexes with the same claudin stoichiometry form pores with unique architecture. Our findings provide insight into tight junction organization and propose a model whereby different claudins combine to form multiple distinct complexes that modify epithelial barrier function by altering tight junction structure.

VX-770 (ivacaftor) is approved for clinical use in CF patients bearing multiple CFTR mutations. VX-770 potentiated wildtype CFTR and several disease mutants expressed in oocytes in a manner modulated by PKA-mediated phosphorylation. Potentiation of some other mutants, including G551D-CFTR, was less dependent upon the level of phosphorylation, likely related to the severe gating defects in these mutants exhibited in part by a shift in PKA sensitivity to activation, possibly due to an electrostatic interaction of D551 with K1250. Phosphorylation-dependent potentiation of wildtype CFTR and other variants also was observed in epithelial cells. Hence, the efficacy of potentiators may be obscured by a ceiling effect when drug screening is performed under strongly phosphorylating conditions. These results should be considered in campaigns for CFTR potentiator discovery, and may enable the expansion of VX-770 to CF patients bearing ultra-orphan CFTR mutations.

Mutations in the bestrophin-1 (Best1) gene are linked to several kinds of macular degeneration in both humans and dogs. Although bestrophins have been shown clearly to be Cl− ion channels, it is controversial whether Cl− channel dysfunction can explain the diseases. It has been suggested that bestrophins are multi-functional proteins: they may regulate voltage-gated Ca2+ channels in addition to functioning as Cl− channels. Here we show that hBest1 differentially modulates Cav1.3 (L-type) voltage-gated Ca2+ channels through association with the Cavβ subunit. In transfected HEK-293 cells, hBest1 inhibited Cav1.3. Inhibition of Cav1.3 was not observed in the absence of the β subunit. Also, the hBest1 C-terminus binds to Cavβ subunits, suggesting that the effect of hBest1 was mediated by the Cavβ subunit. The region of hBest1 responsible for the effect was localized to a region (amino acids 330 − 370) in the cytoplasmic C-terminus that contains a predicted SH3-binding domain that is not present in other bestrophin subtypes. Mutation of Pro330 and Pro334 abolished the effects of hBest1 on Cav1.3. The effect was specific to hBest1: it was not observed with mBest1, -2, or -3. Wild type hBest1 and the disease-causing mutants R92S, G299R, and D312N inhibited Cav currents the same amount, whereas the A146K and G222E mutants were less effective. We propose that hBest1 regulates Cav channels by interacting with the Cavβ subunit and altering channel availability. Our findings reveal a novel function of bestrophin in regulation of Cav channels and suggest a possible mechanism for the role of hBest1 in macular degeneration.

In this paper we present a transistor circuit model for cystic fibrosis transmembrane conductance regulator (CFTR) that seeks to map the functional form of CFTR both in wild type and mutants. The circuit architecture is configured so that the function, and as much as possible the form, faithfully represents what is known about CFTR from cryo-electron microscopy and molecular dynamics. The model is a mixed analog-digital topology with an AND gate receiving the input from two separate ATP-nucleotide-binding domain binding events. The analog portion of the circuit takes the output from the AND gate as its input. The input to the circuit model and its noise characteristics are extracted from single-channel patch-clamp experiments. The chloride current predicted by the model is then compared with single-channel patch-clamp recordings for wild-type CFTR. We also consider the patch-clamp recordings from CFTR with a G551D point mutation, a clinically relevant mutant that is responsive to therapeutic management. Our circuit model approach enables bioengineering approaches to CFTR and allows biophysicists to use efficient circuit simulation tools to analyze its behavior.

Background: Two salt bridges, Arg347–Asp924 and Arg352–Asp993, have been identified in CFTR, but the timing of their interaction remains unknown. Results: Arg347-Asp924-Asp993 form a triangular salt bridge and work together with the Arg352-Asp993 salt bridge to maintain the open pore architecture of CFTR. Conclusion: These salt bridge residues interact and contribute differently in stabilizing the open pore during gating cycle. Significance: Understanding the CFTR pore dynamic open-closed transition is crucial for rational drug design.

The closing of the gated ion channel in the cystic fibrosis transmembrane conductance regulator can be categorized as nonpermissive to reopening, which involves the unbinding of ADP or ATP, or permissive, which does not. Identifying the type of closing is of interest as interactions with nucleotides can be affected in mutants or by introducing agonists. However, all closings are electrically silent and difficult to differentiate. For single-channel patch-clamp traces, we show that the type of the closing can be accurately determined by an inference algorithm implemented on a factor graph, which we demonstrate using both simulated and lab-obtained patch-clamp traces.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily, an ancient family of proteins found in all phyla. In nearly all cases, ABC proteins are transporters that couple the hydrolysis of ATP to the transmembrane movement of substrate via an alternating access mechanism. In contrast, CFTR is best known for its activity as an ATP-dependent chloride channel. We asked why CFTR, which shares the domain architecture of ABC proteins that function as transporters, exhibits functional divergence. We compared CFTR protein sequences to those of other ABC transporters, which identified the ABCC4 proteins as the closest mammalian paralogs, and used statistical analysis of the CFTR-ABCC4 multiple sequence alignment to identify the specific domains and residues most likely to be involved in the evolutionary transition from transporter to channel activity. Among the residues identified as being involved in CFTR functional divergence, by virtue of being both CFTR-specific and conserved among all CFTR orthologs, was R352 in the sixth transmembrane helix (TM6). Patch-clamp experiments show that R352 interacts with D993 in TM9 to stabilize the open-channel state; D993 is absolutely conserved between CFTRs and ABCC4s. These data suggest that CFTR channel activity evolved, at least in part, by converting the conformational changes associated with binding and hydrolysis of ATP, as are found in true ABC Transporters, into an open permeation pathway by means of intraprotein interactions that stabilize the open state. This analysis sets the stage for understanding the evolutionary and functional relationships that make CFTR a unique ABC transporter protein.

Altered cholesterol homeostasis in cystic fibrosis patients has been reported, although controversy remains. As a major membrane lipid component, cholesterol modulates the function of multiple ion channels by complicated mechanisms. However, whether cholesterol directly modulates cystic fibrosis transmembrane conductance regulator (CFTR) channel function remains unknown. To answer this question, we determined the effects of changing plasma membrane cholesterol levels on CFTR channel function utilizing polarized fischer rat thyroid (FRT) cells and primary human bronchial epithelial (HBE) cells. Treatment with methyl-β-cyclodextrin (MβCD) significantly reduced total cholesterol content in FRT cells, which significantly decreased forskolin (FSK)-mediated activation of both wildtype (WT-) and P67L-CFTR. This effect was also seen in HBE cells expressing WT-CFTR. Cholesterol modification by cholesterol oxidase and cholesterol esterase also distinctly affected activation of CFTR by FSK. In addition, alteration of cholesterol increased the potency of VX-770, a clinically used potentiator of CFTR, when both WT- and P67L-CFTR channels were activated at low FSK concentrations; this likely reflects the apparent shift in the sensitivity of WT-CFTR to FSK after alteration of membrane cholesterol. These results demonstrate that changes in the plasma membrane cholesterol level significantly modulate CFTR channel function and consequently may affect sensitivity to clinical therapeutics in CF patients.

Mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator protein (CFTR) cause cystic fibrosis (CF), the most common life-shortening genetic disease among Caucasians. Although general features of the structure of CFTR have been predicted from homology models, the conformational changes that result in channel opening and closing have yet to be resolved. We created new closed- and open-state homology models of CFTR, and performed targeted molecular dynamics simulations of the conformational transitions in a channel opening event. The simulations predict a conformational wave that starts at the nucleotide binding domains and ends with the formation of an open conduction pathway. Changes in side-chain interactions are observed in all major domains of the protein, and experimental confirmation was obtained for a novel intra-protein salt bridge that breaks near the end of the transition. The models and simulation add to our understanding of the mechanism of ATP-dependent gating in this disease-relevant ion channel.

The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) bears six extracellular loops (ECL1–6); ECL1 is the site of several mutations associated with CF. Mutation R117H has been reported to reduce current amplitude, whereas D110H, E116K, and R117C/L/P may impair channel stability. We hypothesized that these amino acids might not be directly involved in ion conduction and permeation but may contribute to stabilizing the outer vestibule architecture in CFTR. We used cRNA injected oocytes combined with electrophysiological techniques to test this hypothesis. Mutants bearing cysteine at these sites were not functionally modified by extracellular MTS reagents and were blocked by GlyH-101 similarly to WT-CFTR. These results suggest that these three residues do not contribute directly to permeation in CFTR. In contrast, mutants D110R-, E116R-, and R117A-CFTR exhibited instability of the open state and significantly shortened burst duration compared with WT-CFTR and failed to be locked into the open state by AMP-PNP (adenosine 5′-(β,γ-imido) triphosphate); charge-retaining mutants showed mainly the full open state with comparably longer open burst duration. These interactions suggest that these ECL1 residues might be involved in maintaining the outer pore architecture of CFTR. A CFTR homology model suggested that E116 interacts with R104 in both the closed and open states, D110 interacts with K892 in the fully closed state, and R117 interacts with E1126 in the open state. These interactions were confirmed experimentally. The results suggest that D110, E116, and R117 may contribute to stabilizing the architecture of the outer pore of CFTR by interactions with other charged residues.