CIBICI   14215
CENTRO DE INVESTIGACION EN BIOQUIMICA CLINICA E INMUNOLOGIA
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Molecular characterization of the I- transport defect-causing Δ287-288 Na+/I- symporter (NIS) mutant uncovers residues involved in Na+ binding/translocation
Autor/es:
NICOLA, JP; REYNA-NEYRA, A; CARRASCO, N
Lugar:
Florianópolis
Reunión:
Congreso; XV Congreso de la Sociedad Latinoamericana de Tiroides; 2013
Institución organizadora:
Sociedad Latinoamericana de Tiroides
Resumen:
Background. Na+/I- symporter (NIS)-mediated active accumulation of I- is a key step in the biosynthesis of thyroid hormones. Several NIS mutations have been identified as a cause of congenital I- transport defect (ITD), and their investigation has yielded valuable mechanistic information on NIS. A NIS mutant lacking amino acids L287 and G288 (Δ287-288) was reported to be non-functional, but was not characterized any further. Objective. To understand the functional impairment caused by the absence of residues 287-288, located in transmembrane segment (TMS) VIII of NIS. Methods. The activity of several TMS VIII NIS mutants was investigated using flux assays (steady state and initial rates), and NIS expression/localization was assessed by immunofluorescence and flow-cytometry in transiently transfected Cos-7 cells. Results. Δ287-288 NIS was intracellularly retained in the ER; therefore, it was not targeted to the plasma membrane and mediated no I- transport. Furthermore, the mutant is intrinsically inactive, as membrane vesicles from cells expressing Δ287-288 NIS did not translocate I-. In addition, Ala substitution at positions 287 and 288 fully recovered NIS plasma membrane targeting and activity. Using double Ala insertions in the Δ287-288 NIS background, we identified the region from N285 to V293 as essential for NIS function. Ala replacements at positions N285, Q286, and L289 yielded NIS proteins with severely reduced apparent affinity for Na+.  Conclusion. Residues N285, Q286, and L289, all of which putatively face the same side of the helix in TMS VIII, play key roles in NIS function and seem to be involved in Na+ binding/translocation.