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.