Study of the epithelial ion transport in physiology and human diseases (cystic fibrosis, Dry Eye Disease) caused by dysfunction of ion channels (channelopathies)

Responsable :  Frédéric BECQ

Based on more than 30 years expertise and know-how our research follows two directions. The first one a better understanding of the molecular basis of epithelial transport physiology (role of chloride channels; calcium homeostasis; regulation and CFTR-related water transport in epithelia). The second one is dedicated to the pharmacology of CFTR, including search for novel modulators (direct binders or proteostasis modulators). Our research leans on specific experimental expertise recognized at the national and international level, such as investigation of ion channels activity and pharmacology and investigation of calcium homeostasis in epithelia or CFTR activity in smooth muscles. For this, we handle multiple experimental procedures:

• Recording and analyzing the activity of ion channels at the unit scale and/or at cellular levels, performed by conventional or robotic patch-clamp devices (Patchliner Nanion);
• Recording transepithelial currents with Ussing chambers measuring short-circuit currents (two set-up for simultaneous recording of 12 tissues)
• Recording in living cells intracellular calcium signals by fluorescence microscopy and fluorescent calcium probes;
• Recording CFTR activity in smooth muscles by ex vivo approach (isolated rings of trachea, bronchus or arteries);
• Recording in living cells CFTR- and AQP-dependent water movement through phase microscopy;
• Measuring in epithelial cells and membranes, lipids in normo or hypoxic situations

 

Chloride channels in human physiology

The transport of ions across cellular membranes is crucial for various functions, including the transport of salt and water across epithelia, the control of electrical excitability of muscle and nerve, and the regulation of cellular volume or the acidification and ionic homeostasis of intracellular organelles. Among the ions transported in living cells, chloride ions (Cl^–) are of particular interest. As Cl^– channels allow only the passive, diffusional flux of Cl^– down its electrochemical gradient, the difference between cytoplasmic and extracellular Cl^– concentration ([Cl^–]), together with the membrane voltage, determines whether the opening of a Cl^– channel lead to an influx or efflux of this ion.

Given this broad range of functions, it is therefore not surprising that mutations in Cl^– channels lead to a large spectrum of diseases. These diverse pathologies include the muscle disorder myotonia, Cystic Fibrosis (CF), renal salt loss in Bartter syndrome, kidney stones, deafness, and the bone disease osteopetrosis. Abnormal regulation of Cl^– channels also lead to water and salt loss in intestinal diseases (e.g. diarrhea). Beside CF, chloride channels are also proposed to play a role in other pulmonary diseases such as asthma and COPD. For these different pathologies, chloride channels modulators (inhibitors, activators/potentiators) constitute an opportunity to develop future medicaments, as it is the case for CF.

 

The problem addressed in Cystic Fibrosis (CF)

CF is caused by mutations in the gene CFTR. The protein product is called Cystic Fibrosis Transmembrane conductance Regulator (CFTR). Physiologically, CFTR operates as a cyclic adenosine monophosphate (cAMP)-regulated and ATP-gated chloride (Cl⁻) channel in the apical membrane of epithelial cells. CFTR plays an essential role in fluid and electrolyte transport across epithelial cells lining ducts and tubes throughout the body. The molecular basis of most cases of CF is temperature-sensitive protein misfolding caused by the deletion of the phenylalanine residue at position 508 of the protein sequence; 90% of CF patients carry at least one copy with F508del mutation. Because misfolded F508del-CFTR is retained in the endoplasmic reticulum at normal body temperature and degraded by the proteasome, the vast majority of F508del-CFTR fails to traffic to the apical membrane of epithelia. The small amount of F508del-CFTR that reaches its correct cellular location forms highly unstable anion channels with a pronounced defect in channel gating. Thus, small-molecules with at least two types of activity are required to restore the plasma membrane expression and function of F508del-CFTR. First, CFTR correctors, so called because they overcome the processing defect of F508del-CFTR and deliver the mutant protein to the apical membrane. Second, CFTR potentiators, so called because they enhance channel gating following CFTR phosphorylation by protein kinase A (PKA).

 

The problem addressed in Dry Eye Disease (DED)

Dry eye (Dry Eye Disease or DED) is a multifactorial and complex disease of the ocular surface leading to a loss of integrity of the tear film inducing various ocular symptoms with potential damage to the ocular surface. DED has a worldwide prevalence higher in women, the elderly and in Asian countries. However, therapeutic strategies are currently limited. Research in this field suffers from a lack of relevant and suitable human in vitro cellular experimental models to study the mechanism of tear secretion and to develop original new therapeutics. Several molecular actors are responsible for making tears, including CFTR and the water carrier Aquaporin (AQP). Recent literature using animal models shows that a therapeutic challenge in DED is to stimulate the activity of these transporters to restore or increase tear volume.                To study the origin of tears and DED we (i) isolate from human samples the epithelial cells involved in the production of the tear film. These cells, once isolated, are amplified and then stored in a biobank. Different experimental models of 2D or 3D cultures are generated to search for agents stimulating tear secretion.