Functional and molecular characterization of pendrin, the anion exchanger involved in Pendred syndrome, non-syndromic deafness and respiratory distresses.

Pendrin is an anion exchanger with affinity for several monovalent anions including iodide, chloride and bicarbonate. It is expressed in the inner ear, thyroid and kidney and more recently was also detected in the airways and in a number of other tissues. Pendrin was first identified as the protein that, if mutated, is responsible for the pathology referred to as Pendred syndrome, an autosomal recessive disorder characterized by bilateral sensorineural hearing loss associated to thyroid dysfunction with or without involvement of the vestibular system. Hypo- or non-functional mutations of pendrin are also responsible for the development of unilateral or bilateral hearing loss with enlargement of the vestibular aqueduct with no involvement of other organs (non-syndromic EVA). On the other hand, also the overexpression and hyper-function of pendrin have recently been associated with a number of other pathologies, such as hypertension, asthma and chronic obstructive pulmonary disease (COPD). In this work, we performed the functional characterization of 7 pendrin variants identified in a cohort of 58 deaf patients by our collaborators of the University of Campinas (Sao Paolo, Brazil), in order to define their role in the observed phenotype. The analysis of pendrin activity was performed by means of a fluorometric assay based on a halide-sensitive enhanced yellow fluorescence protein (EYFP) variant. Furthermore, the molecular defect of such mutants was analyzed, defining their subcellular localization and expression levels by means of confocal microscopy and western blot. We could assess the degree of functional impairment of the analyzed pendrin variants, contributing therefore to the genetic diagnosis of the screened patients. The genetic diagnosis of 5 out of 14 patients was definitively assessed as pendrin-related deafness, while for the remaining patients further genetic investigation is necessary, involving pendrin promoter and 3’-UTR, as well as further deafness related genes. We could observe a clear correlation between measured functional impairment, sub-cellular localization and expression levels of these specific variants. In particular, loss of function variants (T193I and L445W) are retained in the ER and completely excluded from the plasma membrane, while reduction of function mutations (P142L, G149R, C282Y) show at least a partial trafficking to the PM. Based on these findings we hypothesized that pendrin mutant isoforms are targeted for proteasomal degradation. Starting from this hypothesis we could show that pharmacological inhibition of proteasomal degradation with 10µM MG132 was capable to recover (i) total and (ii) plasma membrane expression levels of pendrin variants and, most importantly, (iii) transport activity of specific pendrin variants (P142L, T183I, Q413R, L445W) was improved upon treatment. Such result allows for further development of possible rescuing approaches of pendrin transport activity as a potential treatment of pendrin-related pathologies. A second part of the present study focuses on the search of a specific ligand of pendrin, with the aim of modulating the activity of the transporter, especially in those cases of pathological over-expression and hyperactivity, such as asthma, COPD and hypertension. No specific, non-toxic and potent inhibitor of pendrin could be identified so far and the screening of large compound libraries failed in identifying potential inhibitors. We therefore adopted a more targeted selection of candidates for the screening. We selected compounds among (i) established anion exchangers inhibitors, (ii) inhibitors of the pendrin homologue DRA, (iii) common diuretics and antihypertensive agents, and (iv) via two bioinformatic tools: meta-analysis and SHED alignments. Meta-analysis is an approach aimed to the mathematical definition of a protein topology based on its primary aminoacidic sequence, with no knowledge of its three-dimensional structure. SHED profile allows for the mathematical definition of small compounds according to the intra-molecular distribution of specific atomic features. The effect of the candidates ligands on pendrin activity was evaluated by means of the same fluorometric assay mentioned earlier. In the present study we could (i) confirm the inhibitory effect of previously assessed active compounds, Niflumic acid and NPPB, (ii) define for the first time the inhibitory effect of the anti-inflammatory, anti-rheumatic drug tenidap. In a second screening phase on further compounds suggested by the alignment of the so far identified active compounds niflumic acid and tenidap, according to the SHED profilng, we were able to identify two further active compounds, N-(2-chlorophenyl)-2-(2,4-dibromophenoxy)acetamide and flufenamic acid.