THYROID NA+ I- SYMPORTER - MECHANISM, STOICHIOMETRY, AND SPECIFICITY/

The rat thyroid Na+/l(-) symporter (NIS) was expressed in Xenopus laev is oocytes and characterized using electrophysiological, tracer uptake , and electron microscopic methods, MS activity was found to be electr ogenic and Na+-dependent (Na+ much greater than Li+ much greater than H+). The apparent affinity constants for Na+ and I- were 28 +/- 3 mM a nd 33 +/- 9 mu M, respectively, Stoichiometry of Na+/anion cotransport was 2:1. MS was capable of transporting a wide variety of anions (I-, ClO3-, SCN-, SeCN-, NO3-, Br-, BF4-, IO4-, BrO3-, but perchlorate (Cl O4-) was not transported, In the absence of anion substrate, NIS exhib ited a Na+-dependent leak current (similar to 35% of maximum substrate -induced current) with an apparent Na+ affinity of 74 +/- 14 mM and a Hill coefficient (n) of 1. In response to step voltage changes, MS exh ibited current transients that relaxed with a time constant of 8-14 ms , Presteady-state charge movements (integral of the current transients ) versus voltage relations obey a Boltzmann relation, The voltage for half-maximal charge translocation (V-0.5) was -15 +/- 3 mV, and the ap parent valence of the movable charge was 1. Total charge was insensiti ve to [Na+](o), but V-0.5 shifted to more negative potentials as [Na+] (o) was reduced. MS charge movements are attributed to the conformatio nal changes of the empty transporter within the membrane electric fiel d, The turnover rate of NIS was greater than or equal to 22 s(-1) in t he Na+ uniport mode and greater than or equal to 36 s(-1) in the Na+/I - cotransport mode, Transporter density in the plasma membrane was det ermined using freeze-fracture electron microscopy, Expression of NIS i n oocytes led to a similar to 2,5-fold increase in the density of plas ma membrane protoplasmic face intramembrane particles. On the basis of the kinetic results, we propose an ordered simultaneous transport mec hanism in which the binding of Na+ to NIS occurs first.