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• W2049344067 abstract "The rat thyroid Na+/I− symporter (NIS) was expressed inXenopus laevis oocytes and characterized using electrophysiological, tracer uptake, and electron microscopic methods. NIS activity was found to be electrogenic and Na+-dependent (Na+ ≫ Li+ ≫ H+). The apparent affinity constants for Na+ and I− were 28 ± 3 mm and 33 ± 9 ॖm, respectively. Stoichiometry of Na+/anion cotransport was 2:1. NIS was capable of transporting a wide variety of anions (I−, ClO3−, SCN−, SeCN−, NO3−, Br−, BF4−, IO4−, BrO3−, but perchlorate (ClO4−) was not transported. In the absence of anion substrate, NIS exhibited a Na+-dependent leak current (∼357 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, NIS exhibited 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 apparent valence of the movable charge was 1. Total charge was insensitive to [Na+] o, but V 0.5 shifted to more negative potentials as [Na+] o was reduced. NIS charge movements are attributed to the conformational changes of the empty transporter within the membrane electric field. The turnover rate of NIS was ≥22 s−1 in the Na+ uniport mode and ≥36 s−1 in the Na+/I− cotransport mode. Transporter density in the plasma membrane was determined using freeze-fracture electron microscopy. Expression of NIS in oocytes led to a ∼2.5-fold increase in the density of plasma membrane protoplasmic face intramembrane particles. On the basis of the kinetic results, we propose an ordered simultaneous transport mechanism in which the binding of Na+ to NIS occurs first. The rat thyroid Na+/I− symporter (NIS) was expressed inXenopus laevis oocytes and characterized using electrophysiological, tracer uptake, and electron microscopic methods. NIS activity was found to be electrogenic and Na+-dependent (Na+ ≫ Li+ ≫ H+). The apparent affinity constants for Na+ and I− were 28 ± 3 mm and 33 ± 9 ॖm, respectively. Stoichiometry of Na+/anion cotransport was 2:1. NIS was capable of transporting a wide variety of anions (I−, ClO3−, SCN−, SeCN−, NO3−, Br−, BF4−, IO4−, BrO3−, but perchlorate (ClO4−) was not transported. In the absence of anion substrate, NIS exhibited a Na+-dependent leak current (∼357 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, NIS exhibited 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 apparent valence of the movable charge was 1. Total charge was insensitive to [Na+] o, but V 0.5 shifted to more negative potentials as [Na+] o was reduced. NIS charge movements are attributed to the conformational changes of the empty transporter within the membrane electric field. The turnover rate of NIS was ≥22 s−1 in the Na+ uniport mode and ≥36 s−1 in the Na+/I− cotransport mode. Transporter density in the plasma membrane was determined using freeze-fracture electron microscopy. Expression of NIS in oocytes led to a ∼2.5-fold increase in the density of plasma membrane protoplasmic face intramembrane particles. On the basis of the kinetic results, we propose an ordered simultaneous transport mechanism in which the binding of Na+ to NIS occurs first. It is now firmly established that active accumulation of iodide (I−) by the thyroid gland epithelium, previously referred to as the 舠iodide pump舡 or 舠iodide trap,舡 is a Na+-dependent secondary active transport process mediated by the Na+/I− symporter (NIS), 1The abbreviations used are: NIS, Na+/I− symporter; MES, 2-(N-morpholino)ethanesulfonic acid; FRTL, Fisher rat thyroid line; hPEPT1, human intestinal oligopeptide transporter; IMP, intramembrane particle; n, Hill coefficient; N, sample size; SGLT, Na+/glucose cotransporter; SMIT, Na+/myo-inositol cotransporter. an integral plasma membrane protein of the basolateral membrane of the thyroid gland follicular cells. Iodide transport into the thyroid gland has attracted substantial scientific and clinical interest due to the importance of I− in the biosynthesis of thyroid hormones triiodothyronine and tetraiodothyronine, and to the significance of NIS in the diagnosis and treatment of thyroid disorders (1Carrasco N. Biochim. Biophys. Acta. 1993; 1154: 65-82Crossref PubMed Scopus (343) Google Scholar). A cDNA clone encoding NIS has recently been isolated, sequenced, and expressed in Xenopus laevis oocytes (2Dai G. Levy O. Carrasco N. Nature. 1996; 379: 458-460Crossref PubMed Scopus (970) Google Scholar). Oocytes injected with NIS cRNA exhibit a 700-fold increase in perchlorate-sensitive I− uptake. This study reports a comprehensive characterization of rat NIS function expressed in X. laevis oocytes. NIS activity is Na+-dependent and electrogenic, and the stoichiometry of cotransport is 2 Na+:1 anion. Kinetics of transport as a function of external Na+ and substrate concentration suggest an ordered binding of Na+ and substrate to the transporter in which binding of Na+ occurs first. Substrate selectivity experiments show that a variety of anions are transported into the cell via NIS. However, perchlorate, the most potent known inhibitor of NIS, is not transported. Measurements of charge movements associated with NIS conformational changes, and substrate-uncoupled Na+-dependent leak currents of NIS have provided insight into the nature of Na+/I− cotransport. Combined data from electrophysiological measurements and freeze fracture electron microscopy suggest that NIS may be multimeric in its functional form. NIS cRNA (50 ng) was microinjected into stage V-VI X. laevis oocytes (2Dai G. Levy O. Carrasco N. Nature. 1996; 379: 458-460Crossref PubMed Scopus (970) Google Scholar, 3Parent L. Supplisson S. Loo D.D.F. Wright E.M. J. Membr. Biol. 1992; 125: 49-62Crossref PubMed Scopus (217) Google Scholar), and the oocytes were maintained in Barth's solution at 18 °C until used for experiments. Oocytes were superfused with buffers containing (in mm): 100–0 NaCl, 0–100 choline chloride, 2 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, pH 7.5. Chloride was replaced with gluconate in Cl−-free solutions. For cation selectivity experiments, NaCl was replaced with choline chloride or LiCl at pH 7.5 or choline chloride at pH 5.0 (adjusted with MES). Electrophysiological recordings were done using the two-microelectrode voltage clamp technique at 22 ± 1 °C (4Loo D.D.F. Hazama A. Supplisson S. Turk E. Wright E.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5767-5771Crossref PubMed Scopus (206) Google Scholar). To obtain current/voltage (I/V) or charge/voltage (Q/V) relations, the pulse protocol (pCLAMP, Axon Instruments) consisted of 100-ms voltage steps from a holding potential (V h) of −50 mV to a series of test voltages (V m) from +50 to −150 mV in 20 mV decrements. Currents from three sweeps were averaged, low-pass filtered at 500 Hz, and sampled at 100 ॖs. Uptake of125I− (15 ॖCi/mol; Amersham Corp.) was determined in NIS cRNA-injected oocytes in the presence of 100 mm Na+ and 50 ॖm I−. V h was −90 mV, and substrate-evoked inward currents were recorded for 10 min. Total inward charge movement was determined by integration of the current with time. At the end of the recording period, oocytes were washed with ice-cold choline buffer, solubilized with 107 sodium dodecyl sulfate, and assayed for125I− content. 22Na+(2.5 ॖCi/mol; DuPont) uptake was determined in the presence of 30 mm Na+ (choline, 70 mm) and in the presence or absence of 5 mm anionic substrate. After maximum charge (Q max) measurements (see below), oocytes were fixed as described previously (5Zampighi G.A. Kreman M. Boorer K.J. Loo D.D.F. Bezanilla F. Chandy G. Hall J.E. Wright E.M. J. Membr. Biol. 1995; 148: 65-78Crossref PubMed Scopus (209) Google Scholar). Images of P (protoplasmic) and E (exoplasmic) fracture faces where enlarged to a final magnification of × 75,000 and intramembrane particles (IMP) from both the P and E fracture faces where counted from known areas of the membrane. Total number of transporters per oocyte was estimated by determining the total area of the oocyte plasma membrane from the total plasma membrane capacitance (5Zampighi G.A. Kreman M. Boorer K.J. Loo D.D.F. Bezanilla F. Chandy G. Hall J.E. Wright E.M. J. Membr. Biol. 1995; 148: 65-78Crossref PubMed Scopus (209) Google Scholar), and assuming a membrane specific capacitance of 1 microfarads/cm2. The diameter of the P face IMPs was measured directly from the negative using a profile projector (Nikon, model 6c). Substrate-evoked currents were obtained as the difference in steady-state current measured in the absence and presence of substrate and were fitted to, I=ImaxS·[S]n(K0.5S)n+[S]nEquation 1 where I is the evoked current, Imax S is the maximum current, S is the substrate (anion or Na+), K0.5 S is the substrate concentration at half-maximal current, and n is the Hill coefficient. To obtain the presteady-state currents, total currents were fitted to Equation 2, and transporter-mediated transients were determined by subtracting the capacitive and steady-state components, Itotal(t)=ICmexp(−t/τ1)+IPSexp(−t/τ2)+ISSEquation 2 where I total is the total current,I Cm is the initial membrane capacitive current, τ1 is the time constant of I Cm,I PS is the initial presteady-state current, τ2 is the time constant of I PS, and I SS is the steady-state current. Q/V relations were obtained by integration of the presteady-state current with time for various voltages and were fitted to the Boltzmann relation, Q−QhypQmax=11+exp[z(Vm−V0.5)F/RT]Equation 3 where the total charge Q max =Q dep − Q hyp(Q dep and Q hyp representQ at depolarizing and hyperpolarizing limits), zis the apparent valence of the moveable charge,V m is the membrane voltage during the pulse,V 0.5 is the membrane voltage at which half of the total charge has moved, F is Faraday's constant,R is the gas constant, and T is the absolute temperature. Unless otherwise indicated, results obtained from experiments on individual oocytes are presented, but all experiments were repeated on at least three oocytes from different donor frogs. Data fits were performed using Clampfit (Axon Instruments) or Sigma Plot (Jandel Scientific). Errors are reported as S.E. of the estimate obtained from the fit or as S.E. of the mean obtained from data from several oocytes. Electrogenicity of NIS is shown in Fig.1. Addition of 500 ॖmI− to the bathing medium caused an inward current of ∼400 nA in an oocyte expressing NIS. ClO4− (500 ॖm), a specific blocker of I− transport by the Na+/I− symporter, abolished the I−-evoked inward current. Fig.2 shows typical I/Vrelationships in a NIS-expressing oocyte before (A) and after (B) addition of I− (500 ॖm) to the bath. In the absence of substrate (Fig. 2 A), after the initial fast capacitive transient (τ = 0.5 ms), NIS exhibited slower presteady-state currents that relaxed to a steady state with a time constant of 8–14 ms (see also Fig. 8). Presteady-state currents were apparently abolished by the addition of I− (Fig.2 B). Addition of I− led to a depolarization of the membrane, the magnitude of which depended on the level of NIS expression, and ranged from 5 to 50 mV (not shown).Figure 2Voltage and concentration dependence of I−-induced inward currents. Current traces were obtained from a NIS cRNA-injected oocyte before (A) and after (B) addition of I− (500 ॖm) to the perfusion solution. The pulse protocol is shown. In Athe presteady-state currents associated with NIS are evident (see also Fig. 8). B, addition of I− (500 ॖm) apparently eliminated the presteady-state currents and induced an inward current. Dotted traces show current at the holding potential and emphasize the difference caused by the addition of I−. C, net I−-evoked inward current was taken as the difference between the steady-state current in the presence (1–100 ॖm) and absence of I− and plotted as a function ofV m.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 8Presteady-state currents: charge movement. A, typical current traces of a NIS-expressing oocyte in response to voltage steps from a V h of −50 mV in the absence of substrate. The pulse protocol was as shown in Fig. 2. The current trace for each voltage step was fitted to Equation 2. τ1, the time constant of the membrane capacitive current was ∼0.5 ms for the largest voltage step (100 mV). The dotted line represents zero current at V h. B, carrier-mediated transient currents. The traces in this figure were obtained by subtracting the capacitive and steady-state components (obtained from Equation 2) from the total current (A) and are plotted 1.5 ms after the onset of the voltage step. C, Q/V relation of charge movements. Q was determined by time integration of the carrier-mediated transient currents (B) for the ON (Q ON; •) and OFF (Q OFF; ○) transients. The smooth line is a single Boltzmann fit to the average of Q ON andQ OFF according to Equation 3. Q max = 12 ± 0.6 nanocoulombs,z = 0.9 ± 0.1, V 0.5 = −17 ± 2 mV. D, τ/V relation of the charge transfer. τ was determined from the fit of the total currents in A to Equation 2. τON /V (•) was bell-shaped and the smooth line is a Gaussian fit to τON. τmax was 14 ms, and the voltage at which τmax was observed (V τmax) was −54 ± 2 mV. τOFF was voltage-independent and is shown for +50 mV (○). The time constants of the membrane capacitive currents for the ON (▪) and OFF (□) responses are also shown (∼0.5 ms).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The magnitude of the I−-induced inward current increased with hyperpolarizing potentials, but did not saturate within this voltage range (−150 to −10 mV; Fig. 2 C). Iodide-induced inward current was concentration-dependent and saturable. At each voltage, the net I−-induced current was plotted as a function of [I−] and fitted to Equation 1(n = 1). At −50 mV, the apparent affinity constant of NIS for I−(K 0.5I−) ranged from 15 to 75 ॖm and averaged 33 ± 9 ॖm(N = 7; Fig.3 A). The apparent affinity constant of NIS for iodide (K 0.5I−) was relatively voltage-independent between −150 and −50 mV and increased at potentials more positive than −50 mV (Fig. 3 B). Maximal iodide-induced current (I maxI−) increased in a superlinear fashion as the membrane was driven toward more negative potentials and did not saturate in this voltage range (Fig. 3 C). Fig. 4 shows the relative substrate selectivity of NIS. In this experiment, current was monitored as anions were added (500 ॖm) to the perfusion solution. The best transported substrates were I−, ClO3−, SCN−, SeCN−, and NO3−. SCN− was transported to a significant extent, but ClO4− was not transported at all. The transported anions did not induce an appreciable inward current in H2O-injected oocytes from the same batch (see below). NO2−, HCO3−, acetate, succinate, SO32−, CO32−, S2O32−, and P2O44− were not transported (not shown). The relative apparent affinity of NIS for anions (TableI) were: I− (1.00) ≥ SeCN− (0.87) > SCN− (0.34) > ClO3− (0.12) > NO3− (0.04). The relativeI max values (I maxI− = 1) ranged from 0.8 to 1.5 (Table I). The voltage dependence of K 0.5and I max was the same for all of the above anions (not shown).Table IKinetics of anion cotransportAnionK 0.5I maxॖmI−33 ± 91.00SeCN−38 ± 150.8 ± 0.1SCN−96 ± 90.8 ± 0.2ClO3−277 ± 201.0 ± 0.2NO3−739 ± 2231.5 ± 0.2I maxsubstrate values have been normalized toI maxI− obtained from the same oocyte from which the substrate-induced current was obtained. Values are mean ± S.E. from at least three oocytes. Reported values are for V m = −50 mV. Open table in a new tab I maxsubstrate values have been normalized toI maxI− obtained from the same oocyte from which the substrate-induced current was obtained. Values are mean ± S.E. from at least three oocytes. Reported values are for V m = −50 mV. ClO4− completely blocked the current generated by 50 ॖm I−, with an apparent half-inhibition constant (K i ClO4−) of 1.8 ± 0.4 ॖm (N = 4). K i ReO4−was also very low (3.2 ± 0.4 ॖm; N= 5), but this inhibitor could only block the I−-induced current by 86 ± 37. This could be due to the fact that at high concentrations (500 ॖm), ReO4− itself can induce a very small inward current (Fig. 4). BF4− and IO4− (500 ॖm) reduced the I−-evoked (50 ॖm) inward currents by 74 ± 107 and 21 ± 67, respectively (N = 3). In some control (non-injected) oocytes, I− induced a small but detectable inward current, which was prominent at high iodide concentrations (>500 ॖm) and at depolarizing membrane potentials (−10 to +50 mV). These iodide currents were insensitive to perchlorate. Of the anions that are readily transported by NIS (I−, ClO3−, SCN−, SeCN−, and NO3−), only ClO3− did not exhibit this behavior. Therefore, we chose to use ClO3− as a model anion for further kinetic studies. When Na+ in the perfusion solution was isotonically replaced with choline, no I−-induced (500 ॖm) inward current was observed at either pH 7.5 or 5.0. Li+, however, was able to drive transport at a reduced level. At −150 mV, the Li+/I− current was 10–207 of the Na+/I− current (not shown). In Fig.5 A, inward currents induced by 0.25, 1, and 5 mm ClO3− are plotted as a function of external Na+ concentration ([Na+] o). At each substrate concentration, inward currents saturated with increasing [Na+] o. The Hill coefficient for Na+ was ∼2 regardless of the substrate concentration and V m; at [ClO3−] = 1 mm andV m = −50 mV, n = 2.2 ± 0.1. I maxNa+ increased with increasing substrate concentration (Fig. 5 B) and the apparent affinity of NIS for Na+ increased as [ClO3−] was increased (Fig.5 C). At −50 mV, at [ClO3−] = 0.25, 1, and 5 mm, K 0.5Na+ was 57 ± 7, 39 ± 3, and 28 ± 3 mm, respectively. Examination of substrate kinetics at different [Na+] o (Fig.6 A) showed that althoughI maxClO3−remained constant as [Na+] o was lowered (Fig.6 B), the apparent affinity of NIS for substrate decreased dramatically (Fig. 6 C);K 0.5ClO3−was 271 ± 5 ॖm at 100 mm[Na+] o and 1671 ± 263 ॖm at 20 mm [Na+] o. Fig.7 A shows the voltage-dependence ofK 0.5ClO3−at various [Na+] o. Regardless of [Na+] o,K 0.5ClO3−approached 150 ॖm at hyperpolarizing limits. At less negative membrane potentialsK 0.5ClO3−varied greatly depending on [Na+] o. As withK 0.5ClO3−,K 0.5Na+ varied with voltage and with the concentration of cosubstrate (Fig. 7 B). In the absence of substrate, NIS cRNA-injected oocytes exhibited presteady-state current transients in response to step changes in V m (Figs.2 A and 8 A). These current transients were not observed in control H2O-injected oocytes (see Ref. 3Parent L. Supplisson S. Loo D.D.F. Wright E.M. J. Membr. Biol. 1992; 125: 49-62Crossref PubMed Scopus (217) Google Scholar). Fitting of the current traces (both ON and OFF; Fig.8 A) to Equation 2 resolved three components: (i) a fast component (τ ∼0.5 ms) due to oocyte membrane capacitive currents (also seen in control oocytes); (ii) a second slower component (τ ≈8–14 ms), which was the NIS-mediated current; and (iii) a steady-state current due to 舠leak舡 pathways in NIS and the membrane. To obtain the carrier-mediated transients, the membrane capacitive and steady-state components were subtracted from the total current (Fig. 8 B). At each clamped voltage, integration of the carrier-mediated currents (Fig. 8 B) with time yielded the charge (Q) moved by NIS within the membrane electric field. Fig. 8 C shows a Q/V relationship for NIS. Q ON and Q OFFwere equal and opposite in sign and reversed atV h (−50 mV). The Q/V curve fitted a single Boltzmann relation (Equation 3) with aV 0.5 of −15 ± 3 mV and a z of 0.9 ± 0.1 (N = 8). The time constant of the slow current transient was voltage-dependent for the ON response. τON /V was bell-shaped and ranged from 8 to 14 ms with its maximum value at ∼−55 mV (V τmax) (Fig. 8 D). τOFF was voltage-independent at ∼10 ms (○; Fig.8 D). Fig. 9 A shows Q/Vcurves at 0–100 mm [Na+] o. There was no loss in Q max as [Na+] o was reduced from 100–20 mm (Fig. 9 B), butV 0.5 shifted from −17 mV at 100 mm[Na+] o to −90 mV at 20 mm[Na+] o (Fig. 9 C). z was ∼1 at all Na+ concentrations. The maximum value of the time constant of the relaxation currents (τmax ≈14 ms) did not change as [Na+] o was reduced (not shown), but V τmax shifted from ∼−55 mV at 100 mm [Na+] o to ∼−74 mV at 20 mm [Na+] o (not shown). Addition of either substrate or inhibitor led to a reduction inQ max (Fig.10 A). As the concentration of substrate or inhibitor was increased, the decrease inQ max followed a hyperbolic function (not shown). With ClO3−, 50 percent reduction inQ max was reached at 586 ± 80 ॖm (N = 3). The ClO3−-induced reduction inQ max was directly proportional to the steady-state ClO3−-induced inward current (Fig. 10 B). At −50 and −150 mV, the slope of the plot I versus Q was 36 ± 2 s−1 and 61 ± 4 s−1, respectively. In NIS-expressing oocytes, replacement of 100 mm choline chloride with NaCl caused an inward current that was much larger (>100 nA; Fig.11 A) than that seen in H2O-injected oocytes (<20 nA at −50 mV). Addition of 500 ॖm I− caused a further increase in the inward current. The Na+-dependent inward current, in the absence of substrate, is referred to as the NIS Na+ leak current. The Na+ leak current was saturable with increasing [Na+] o. At −50 mV, the [Na+] o at which the leak current was half-maximal ( K0.5leak) was 74 ± 14 mm (N = 3; Fig. 11 B), and the Hill coefficient was 0.9 ± 0.1 (N = 3). Control H2O-injected oocytes exhibited inward Na+currents that had a half-saturation constant of 9 ± 2 mm (N = 3). The magnitude of the leak current increased linearly with the level of expression, such that there was a direct linear correlation between the leak current and the substrate-induced current. At −150 mV, the plot of maximum leak current as a function of maximum ClO3−-induced current ( Imaxleak versus I maxClO−3) yielded a slope of 0.34 ± 0.04 (N = 7; not shown). Fig.12 A shows a current record from a NIS-expressing oocyte held at −90 mV and perfused with a solution containing I− (100 mmNa+, 50 ॖm I−, and 15 ॖCi/mol125I−) for 10 min. Integration of the I−-evoked inward current yielded the net positive charge that entered the oocyte during the recording period (shaded region). In the same oocyte, I− uptake was measured by determination of 125I− content. A plot of the net inward charge versus I− uptake from 10 oocytes revealed a linear relation with a slope of 0.76 ± 0.03 inward charge per iodide uptake (Fig. 12 B). In Fig.12 C, inward charge is plotted as a function of Na+ uptake. Inward current was induced by 5 mmClO3− for 10 min in the presence of 30 mm Na+ and 2.5 ॖCi/mol22Na+. The slope of the line was 0.42 ± 0.04 inward charge per Na+ uptake (N = 6). Freeze-fracture electron micrographs of the P fracture face from the plasma membrane of a control H2O-injected oocyte and an oocyte expressing NIS are shown in Fig. 13. In the control oocyte, the density of IMPs in the P face was 356 ± 69/ॖm2 (mean ± S.D.; Fig. 13 A). The endogenous intramembrane particles showed a relatively homogenous distribution with a mean diameter of 7.6 ± 1.2 nm (N = 875). Oocytes expressing NIS showed a ∼2.5-fold increase in the density of P face particles to 887 ± 146/ॖm2 (Fig. 13 B). In contrast, the density of IMPs in the E face was not altered by expression of NIS (not shown; see Ref. 5Zampighi G.A. Kreman M. Boorer K.J. Loo D.D.F. Bezanilla F. Chandy G. Hall J.E. Wright E.M. J. Membr. Biol. 1995; 148: 65-78Crossref PubMed Scopus (209) Google Scholar). In addition, P face intramembrane particles of NIS-expressing oocytes showed a greater heterogeneity in size. Analysis of the diameter of P face IMPs (N = 856) in oocytes expressing NIS showed two prominent populations: one at 7.2 ± 0.5 nm corresponding to the endogenous particles and another at 9.0 ± 0.6 nm due to NIS particles. In the oocyte in Fig. 13 B,Q max was 18 nanocoulombs and the total number of transporters (N NIS) in the plasma membrane was 3.5 × 1010. Q max =N NIS Ze, where Z is the valence of the moveable charge per NIS particle, and e is the electronic charge. Therefore, Z was estimated to be ∼3 electronic charges. Cloning of the Na+/I− symporter and its expression inX. laevis oocytes has made it possible to carry out a thorough functional characterization of this transporter. Iodide transport via NIS generates a net influx of positive charge (an inward current) that depolarizes the membrane. The inward current is Na+-dependent, stimulated by I−, and coupled to Na+ and I− influx. Uptake studies indicate that 2 Na+ ions are transported with one anion, resulting in inward movement of one positive charge. Previously, the electrogenic nature of the Na+/I−symporter had been suggested in experiments using plasma membrane vesicles from hog thyroid (6O'Neill B. Magnolato D. Semenza G. Biochim. Biophys. Acta. 1987; 896: 263-274Crossref PubMed Scopus (42) Google Scholar). The apparent affinity constant of NIS for I− (33 ± 9 ॖm at −50 mV) is in general agreement with those obtained in uptake studies in NIS-expressing X. laevisoocytes (36 ॖm) (2Dai G. Levy O. Carrasco N. Nature. 1996; 379: 458-460Crossref PubMed Scopus (970) Google Scholar), FRTL-5 cells (30 ॖm) (7Weiss S.J. Philp N.J. Grollman E.F. Endocrinology. 1984; 114: 1108-1113Crossref PubMed Scopus (116) Google Scholar), and membrane vesicles derived form porcine thyroid (5 ॖm) (6O'Neill B. Magnolato D. Semenza G. Biochim. Biophys. Acta. 1987; 896: 263-274Crossref PubMed Scopus (42) Google Scholar, 8Nakamura Y. Ohtaki S. Yamazaki I. J. Biochem. (Tokyo). 1988; 104: 544-549Crossref PubMed Scopus (17) Google Scholar). It is significant to note that the reported free iodide concentration in the mammalian plasma is 50–300 nm (1Carrasco N. Biochim. Biophys. Acta. 1993; 1154: 65-82Crossref PubMed Scopus (343) Google Scholar), while theK 0.5I− determined in this and other studies is in the low micromolar range. The apparent affinity constant of NIS for Na+ (28 ± 3 mm at −50 mV and saturating substrate) is also comparable with that found in other studies (∼50 mm) (6O'Neill B. Magnolato D. Semenza G. Biochim. Biophys. Acta. 1987; 896: 263-274Crossref PubMed Scopus (42) Google Scholar, 9Kaminsky S.M. Levy O. Garry M.T. Carrasco N. Eur. J. Biochem. 1991; 200: 203-207Crossref PubMed Scopus (22) Google Scholar). In addition to I−, a number of other anions are transported by NIS: I− ≥ SeCN− > SCN− > ClO3− > NO3−. The only apparent common denominator for the well transported substrates is anionic monovalency. The closer the size of the monovalent anion to that of I−, the better it is transported (10Halmi N.S. Vitam. Horm. 1961; 19: 133-163Crossref Scopus (51) Google Scholar). No conclusion, however, can be drawn regarding the molecular geometry of a good substrate. Iodide is nearly spherical while SeCN− and SCN− are near-linear; ClO3− has a trigonal pyramidal geometry; and NO3− is planar. Regardless of the geometry of the anion, the qualitative similarity of transport kinetics of I−, SeCN−, SCN−, ClO3−, and NO3− suggests that their mechanism of transport may be the same. A number of anions can significantly inhibit I− transport. Most notable is ClO4−, the most potent known inhibitor of NIS (K i ClO4−= 1.8 ± 0.4 ॖm). Previous reports suggested that ReO4− (perrhenate) is transported into the thyroid (10Halmi N.S. Vitam. Horm. 1961; 19: 133-163Crossref Scopus (51) Google Scholar). Our results show that ReO4− is also a very potent blocker (K i ReO4−= 3.2 ± 0.4 ॖm), but at high concentrations (>500 ॖm) it is transported via NIS to a very small extent (Fig. 4). ClO4− and SCN−were traditionally used as competitive inhibitors of I−uptake in the thyroid gland, and both were believed to be transported via the Na+/I− cotransport system (1Carrasco N. Biochim. Biophys. Acta. 1993; 1154: 65-82Crossref PubMed Scopus (343) Google Scholar, 11Anbar M. Guttmann S. Lewitus Z. J. Appl. Radiat. Isot. 1959; 7: 87-96Crossref PubMed Scopus (42) Google Scholar). In our system, SCN− is transported, but perchlorate is not. That perchlorate is not transported by NIS is not unique to theXenopus oocyte expression system as similar results have been obtained with rat NIS expressed in Chinese hamster ovary cells (12Yoshida A. Sasaki N. Mori A. Taniguchi S. Mitani Y. Ueta Y. Hattori K. Sato R. Hisatome I. Mori T. Shigemasa C. Kosugi S. Biochem. Biophys. Res. Commun. 1997; 231: 731-734Crossref PubMed Scopus (45) Google Scholar). Our data, however, cannot exclude electroneutral Na+/ClO4− transport (1:1 coupling ratio). Thus, anions that effectively interact with NIS can be subdivided into three groups: (i) anions that are readily transported; e.g.I−, SeCN−, SCN−, ClO3−, and NO3−; (ii) anions that partially inhibit I− transport, but are themselves transported to some extent; e.g. IO4−, BF4−, and ReO4−; and (iii) anions that completely inhibit transport; ClO4−. Although no conclusion can be drawn about the molecular commonality of the first group, anions belonging to the second and third groups all have a tetrahedral molecular geometry with an anionic volume very similar to that of I− (13Wolf J. Physiol. Rev. 1964; 44: 45-90Crossref PubMed Scopus (253) Go" @default.
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