The discovery of ClC proteins at the beginning of the 1990s was important for the development of the Cl- transport research field. ClCs form a large family of proteins that mediate voltage-dependent transport of Cl- ions across cell membranes. They are expressed in both plasma and intracellular membranes of cells from almost all living organisms. ClC proteins form transmembrane dimers, in which each monomer displays independent ion conductance. Eukaryotic members also possess a large cytoplasmic domain containing two CBS domains, which are involved in transport modulation. ClC proteins function as either Cl- channels or Cl-/H+ exchangers, although all ClC proteins share the same basic architecture. ClC channels have two gating mechanisms: a relatively well-studied fast gating mechanism, and a slow gating mechanism, which is poorly defined. ClCs are involved in a wide range of physiological processes, including regulation of resting membrane potential in skeletal muscle, facilitation of transepithelial Cl- reabsorption in kidneys, and control of pH and Cl- concentration in intracellular compartments through coupled Cl-/H+ exchange mechanisms. Several inherited diseases result from C1C gene mutations, including myotonia congenita, Bartter’s syndrome (types 3 and 4), Dent’s disease, osteopetrosis, retinal degeneration, and lysosomal storage diseases. This review summarizes general features, known or suspected, of ClC structure, gating and physiological functions. We also discuss biophysical properties of mammalian ClCs that are directly involved in the pathophysiology of several human inherited disorders, or that induce interesting phenotypes in animal models.

Vasoactive intestinal peptide (VIP), a neuropeptide, controls multiple functions in exocrine tissues, including inflammation, and relaxation of airway and vascular smooth muscles, and regulates CFTR-dependent secretion, which contributes to mucus hydration and local innate defense of the lung. We had previously reported that VIP stimulates the VPAC1 receptor, PKCϵ signaling cascade, and increases CFTR stability and function at the apical membrane of airway epithelial cells by reducing its internalization rate. Moreover, prolonged VIP stimulation corrects the molecular defects associated with F508del, the most common CFTR mutation responsible for the genetic disease cystic fibrosis. In the present study, we have examined the impact of the absence of VIP on CFTR maturation, cellular localization, and function in vivo using VIP knockout mice. We have conducted pathological assessments and detected signs of lung and intestinal disease. Immunodetection methods have shown that the absence of VIP results in CFTR intracellular retention despite normal expression and maturation levels. A subsequent loss of CFTR-dependent chloride current was measured in functional assays with Ussing chamber analysis of the small intestine ex vivo, creating a cystic fibrosis-like condition. Interestingly, intraperitoneal administration of VIP corrected tissue abnormalities, close to the wild-type phenotype, as well as associated defects in the vital CFTR protein. The results show in vivo a primary role for VIP chronic exposure in CFTR membrane stability and function and confirm in vitro data.

The CFTR chloride channel is regulated by phosphorylation at PKA and PKC consensus sites within its regulatory region (R-region) through a mechanism, which is still not completely understood. We used a split-CFTR construct expressing the N-term-TMD1-NBD1 (Front Half; FH), TMD2-NBD2-C-Term (Back Half; BH), and the R-region as separate polypeptides (Split-R) in BHK cells, to investigate in situ how different phosphorylation conditions affect the R-region interactions with other parts of the protein. In proximity ligation assays, we studied the formation of complexes between the R-region and each half of the Split-CFTR. We found that at basal conditions, the density of complexes formed between the R-region and both halves of the split channel were equal. PKC stimulation alone had no effect, whereas PKA stimulation induced the formation of more complexes between the R-region and both halves compared to basal conditions. Moreover, PKC + PKA stimulation further enhanced the formation of FH-R complexes by 40% from PKA level. In cells expressing the Split-R with the two inhibitory PKC sites on the R-region inactivated (SR-S641A/T682A), density of FH-R complexes was much higher than in Split-R WT expressing cells after PKC or PKC + PKA stimulation. No differences were observed for BH-R complexes measured at all phosphorylation conditions. Since full-length CFTR channels display large functional responses to PKC + PKA in WT and S641A/T682A mutant, we conclude that FH-R interactions are important for CFTR function. Inactivation of consensus PKC site serine 686 (S686A) significantly reduced the basal BH-R interaction and prevented the PKC enhancing effect on CFTR function and FH-R interaction. The phospho-mimetic mutation (S686D) restored basal BH-R interaction and the PKC enhancing effect on CFTR function with enhanced FH-R interaction. As the channel function is mainly stimulated by PKA phosphorylation of the R-region, and this response is known to be enhanced 34 |