ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Endocrine Abstracts (2008) 16 P770

New mutations demonstrate intracellular iodine retention in Pendred syndrome

MER Garcia-Rendueles1, SB Bravo1, F Palos2, J Cameselle-Teijeiro3, B Czarnocka4, L Dominguez-Gerpe2, J Lado-Abeal2 & CV Alvarez1

1Department of Physiology, University of Santiago de Compostela, Santiago de Compostela, Spain; 2Unidade de Enfermedades Tiroideas e Metabólicas (UETeM), Department of Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain; 3Department of Pathology, Complejo Hospitalario Universitario de Santiago (CHUS)-SERGAS, Santiago de Compostela, Spain; 4Department of Biochemistry, Medical Centre for Postgraduate Education, Warsaw, Poland.

Pendred syndrome is an autosomal recessive disorder with congenital-sensorineural deafness and goiter due to mutations in SLC26A4, that encodes a transmembrane protein, Pendrin. In thyrocytes, Pendrin is proposed to act at the apical pole to transport intracellular iodide into the follicular lumen.

A Galician Pendred compound heterozygous patient was studied; a c.297delT in exon 3 and a new splicing-mutation c.416-1G<A were found, introducing premature stop codons in the protein.

A thyrocyte primary cell-line, T-PS2, was obtained from the patient and compared with primary thyroid lines from our BANTTIC (Bravo Oncogene 2003, Bravo Clin Cancer Res 2005). NT (normal thyrocytes) and T-PS2 have similar epithelial appearance, with follicle-like structures and expressed thyroglobulin.

By western-blot, NT and T-PS2 showed similar levels of plasma-membrane NIS (Na+/I symporter), while only NT showed high levels of plasma-membrane pendrin. Confocal immunofluorescence localized Pendrin in NT at a spot near the nucleus, Golgi location, and in narrow lines typical of plasma-membrane localization. Opposite, in T-PS2 Pendrin positivity was located exclusively in Golgi, indicating retention of truncated proteins.

Iodine-uptake measurements were performed. Steady-state uptake was 3-times higher in T-PS2 than in NT. Time-course uptake in NT showed a fast uptake followed by a plateau after 5 min onward; T-PS2 showed a progressive increase in iodide level till 30 min; Vmax was twice as high in T-PS2.

Efflux was fast for NT: at 15 min all radioactivity had effluxed while 40% still remained intracellularly in T-PS2.

We used dose–response curves to study Michaelis-Menten iodine-uptake kinetics. After 5 min, T-PS2 had already reached a Km similar to that described for NIS at equilibrium (22±4.8 μM). Opposite, NT only achieved equilibrium after 1 h of incubation.

In summary, normal thyrocytes behave as a complex system with two opposite transporters (NIS and Pendrin) that reach equilibrium slowly. Pendred thyrocytes accumulate iodine through NIS, although iodine leaves the cell inefficiently through other non-specific transporters.

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