The Role of the R-domain in Regulated Trafficking of the Cystic Fibrosis Transmembrane Conductance Regulator.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation-regulated chloride channel that is a member of the ATP-binding cassette (ABC) transporter family [1]. It is involved in the movement of chloride ions across epithelial membranes in the airways, sweat glands, intestine and pancreas [2]. Mutations in CFTR that result in a loss of channel function result in the disease cystic fibrosis, affecting nearly 1 in 2500 people in northern Europe and the United States [3]. As a member of the ABC transporter family, CFTR shares the structural features of these proteins. Unique to CFTR is the presence of a cytoplasmic R-domain, that contains multiple phosphorylation sites. Phosphorylation of the R domain is required for CFTR channel gating, and cAMP/PKA simulation can also elicit insertion of CFTR into the plasma membrane from intracellular compartments [4]. We evaluated the structural basis of regulated CFTR trafficking by determining agonist-evoked increases in plasma membrane capacitance (Cm) of Xenopus oocytes expressing CFTR deletion mutants. Expression of CFTR as a split construct that omitted the R-domain (ƒ´aa 635-834) produced a channel with elevated basal current (Im) and no ƒ´Im or trafficking response (ƒ´Cm) upon cAMP/PKA stimulation, indicating that the structure(s) required for regulated CFTR trafficking are contained within the R domain. Additional deletions showed that removal of amino acids 817-838 produced a channel with regulated gating that lacked the agonist-induced increase in CFTR trafficking. This 22aa region exhibits helical structure, bears a net negative charge of -9, is highly conserved among species, and has been termed NEG2 [5, 6]. Injection of NEG2 peptide into oocytes expressing split ƒ´NEG2 CFTR prior to stimulation restored the agonist-evoked ƒ´Cm, consistent with the concept that this sequence mediates regulated CFTR trafficking. Further modifications of NEG2 suggest that the trafficking phenotype depends primarily on its helical structure. These observations suggest that the NEG2 region at the C-terminus of the R domain allows CFTR to enter a regulated intracellular compartment from which it traffics to the plasma membrane in response to cAMP/PKA-stimulation

Functional Interaction between CFTR and the Sodium-Phosphate Co-Transport Type 2a in Xenopus laevis Oocytes

A growing number of proteins, including ion transporters, have been shown to interact with Cystic Fibrosis Transmembrane conductance Regulator (CFTR). CFTR is an epithelial chloride channel that is involved in Cystic Fibrosis (CF) when mutated; thus a better knowledge of its functional interactome may help to understand the pathophysiology of this complex disease. In the present study, we investigated if CFTR and the sodium-phosphate co-transporter type 2a (NPT2a) functionally interact after heterologous expression of both proteins in Xenopus laevis oocytes.NPT2a was expressed alone or in combination with CFTR in X. laevis oocytes. Using the two-electrode voltage-clamp technique, the inorganic phosphate-induced current (IPi) was measured and taken as an index of NPT2a activity. The maximal IPi for NPT2a substrates was reduced when CFTR was co-expressed with NPT2a, suggesting a decrease in its expression at the oolemna. This was consistent with Western blot analysis showing reduced NPT2a plasma membrane expression in oocytes co-expressing both proteins, whereas NPT2a protein level in total cell lysate was the same in NPT2a- and NPT2a+CFTR-oocytes. In NPT2a+CFTR- but not in NPT2a-oocytes, IPi and NPT2a surface expression were increased upon PKA stimulation, whereas stimulation of Exchange Protein directly Activated by cAMP (EPAC) had no effect. When NPT2a-oocytes were injected with NEG2, a short amino-acid sequence from the CFTR regulatory domain that regulates PKA-dependent CFTR trafficking to the plasma membrane, IPi values and NPT2a membrane expression were diminished, and could be enhanced by PKA stimulation, thereby mimicking the effects of CFTR co-expression.We conclude that when both CFTR and NPT2a are expressed in X. laevis oocytes, CFTR confers to NPT2a a cAMPi-dependent trafficking to the membrane. This functional interaction raises the hypothesis that CFTR may play a role in phosphate homeostasis

Toxin Mediated Diarrhea in the 21st Century: The Pathophysiology of Intestinal Ion Transport in the Course of ETEC, V. cholerae and Rotavirus Infection

An estimated 4 billion episodes of diarrhea occur each year. As a result, 2–3 million children and 0.5–1 million adults succumb to the consequences of this major healthcare concern. The majority of these deaths can be attributed to toxin mediated diarrhea by infectious agents, such as E. coli, V. cholerae or Rotavirus. Our understanding of the pathophysiological processes underlying these infectious diseases has notably improved over the last years. This review will focus on the cellular mechanism of action of the most common enterotoxins and the latest specific therapeutic approaches that have been developed to contain their lethal effects

Characterization of SLC26A9 in Patients with CF-Like Lung Disease

International audienceDiffuse bronchiectasis is a common problemin respiratory clinics. We hypothesized that mutations inthe solute carrier 26A9 (SLC26A9) gene, encoding for achloride (Cl−) transporter mainly expressed in lungs, maylead to defects in mucociliary clearance. We describe twomissense variants in theSLC26A9gene in heterozygotepatients presenting with diffuse idiopathic bronchiectasis: p.Arg575Trp, identified in a patient also heterozygotefor p.Phe508del in theCFTRgene; and p.Val486Ile.Expression of both mutants inXenopus laevisoocytesabolished SLC26A9-mediated Cl−conductance withoutdecreasing protein membrane expression. Coexpressionof CFTR with SLC26A9–p.Val486Ile resulted in a sig-nificant increase in the Cl−current induced by PKAstimulation, similar to that obtained in oocytes express-ing CFTR and SLC26A9–WT. In contrast, coexpres-sion of CFTR with SLC26A9–p.Arg575Trp inhibitedSLC26A9-enhanced CFTR activation upon PKA. Fur-ther structure–function analyses led us to propose a siteencompassing Arg575 in the SLC26A9–STAS domainfor CFTR–SLC26A9 interaction. We hypothesize thatSLC26A9–p.Arg575Trp prevented SLC26A9-mediatedfunctional activation of CFTR by altering SLC26A9–CFTR interaction. Although we cannot confirm that thesemutations by themselves are deleterious, we propose thatthey trigger the pathogenic role of a singleCFTRmuta-tion and provide insight into a novel mechanism of Cl−transport alteration across the respiratory mucosa, basedon functional inhibition of CFT