IL TRASPORTO DI CLORURO: COINVOLGIMENTO IN ALCUNE PATOLOGIE UMANE

Several human inherited diseases are caused by mutations in chloride channels or transporters, which cause symptoms as diverse as epilepsy, startle disease, deafness, blindness, lysosomal storage and neurodegeneration, osteopetrosis, lung infections and fibrosis, male infertility, renal salt loss, and kidney stones, clearly indicating the crucial importance of anion transport in many tissues. ICln is a water-soluble protein forming a PH domain, which can be introduced into the cellular membrane to form ion channels. When expressed in cellular systems, wt-ICln ion channels mediate a chloride current resembling those activated after cell swelling (ICl,swell), suggesting a role for ICln in cell volume regulation. During the first part of my PhD program at the department of Pharmacology and Toxicology (Paracelsus Medical University of Salzburg, Austria) I was involved in the functional characterization (by Black Lipid Bilayer and Patch Clamp experiments) of a new mutant form of the ICln protein possibly implicated in a heart disease. The object of my investigation was the insertion of a Thymine in position 383 in the nucleotide sequence. This mutation produces a frameshift, leading to a scrambled sequence of 12 aminoacids (starting from the aminoacid 128) and (an earlier termination (A128FSX139) of the channel sequence. This mutation has been identified in a patient whose familiarity, symptoms and QT value suggested she was suffering from long QT syndrome (LQTS). Black lipid Bilayer experiments showed that the mutated protein reconstituted in artificial membrane of sphingomyelin had the same electrophysiological characteristics of the wild type protein. Otherwise Patch Clamp experiments (whole cell configuration) showed that the overexpression of the mutated protein in HEK293 Phoenix cells produced a significantly reduced ICl,swell current in comparison to that obtained by hIClnWT overexpression. During the second part of my PhD program I was involved in the functional characterization of the 5`flanking region of SLC26A4. SLC26A4 (Pendrin) was cloned by positional characterization of the gene for the Pendred syndrome (OMIM #274600), a recessively inherited disorder causing congenital deafness and thyroid goiter accountable for up to 10% of inherited hearing loss. The purpose of this part of my work was to identify the minimum sequence necessary for the transcription of the SLC26A4 gene and, when possible, to identify sequences important for the “tissue specific“ expression of the gene and for the recognition of responsive elements in a promoter sequence. The experiments performed in both HEK 293 Phoenix cells and in rat thyroid PC-CL3 cells showed that a sequence of only 286 base pairs is able to drive the basal expression of the pendrin gene in both cellular models. We assumed that the sequence contains all the cis-acting signals necessary for the activity of the basal transcription machinery. Moreover experiment performed in PC-CL3 cells allowed us identifying a tireoglobuline (TG) responsive element of only 205 base pairs in the promoter sequence of pendrin protein. Through acquiring an in-depth knowledge of the mechanisms involved in SLC26A4 gene regulation it would be possible to intervene on pendrin expression in case of an anomalous regulation in order to improve symptoms found in patients suffering from PS.