Iodide (I-) is an essential constituent of the thyroid hormones T3 and T4, and is accumulated by the thyroid. The transport of iodide, the first step in thyroid hormogenesis, is catalysed by the Na+/I- symporter, an intrinsic membrane protein that is crucial for the evaluation, diagnosis and treatment of thyroid disorders. Although several other important thyroid proteins involved in hormogenesis have been characterized, the Na+/I- symporter has not. Here we report the isolation of a complementary DNA clone that encodes this symporter, as a result of functional screening of a cDNA library from a rat thyroid-derived cell line (FRTL-5) in Xenopus laevis oocytes. Oocyte microinjection of an RNA transcript made in vitro from this cDNA clone elicited a more than 700-fold increase in perchlorate-sensitive Na+/I- symport activity over background. To our knowledge, this is the first iodide-transporting molecule to have its cDNA cloned, providing a missing link in the thyroid hormone biosynthetic pathway.

Two peptides, ProTx-I and ProTx-II, from the venom of the tarantula Thrixopelma pruriens, have been isolated and characterized. These peptides were purified on the basis of their ability to reversibly inhibit the tetrodotoxin-resistant Na channel, Na(V) 1.8, and are shown to belong to the inhibitory cystine knot (ICK) family of peptide toxins interacting with voltage-gated ion channels. The family has several hallmarks: cystine bridge connectivity, mechanism of channel inhibition, and promiscuity across channels within and across channel families. The cystine bridge connectivity of ProTx-II is very similar to that of other members of this family, i.e., C(2) to C(16), C(9) to C(21), and C(15) to C(25). These peptides are the first high-affinity ligands for tetrodotoxin-resistant peripheral nerve Na(V) channels, but also inhibit other Na(V) channels (IC(50)'s < 100 nM). ProTx-I and ProTx-II shift the voltage dependence of activation of Na(V) 1.5 to more positive voltages, similar to other gating-modifier ICK family members. ProTx-I also shifts the voltage dependence of activation of Ca(V) 3.1 (alpha(1G), T-type, IC(50) = 50 nM) without affecting the voltage dependence of inactivation. To enable further structural and functional studies, synthetic ProTx-II was made; it adopts the same structure and has the same functional properties as the native peptide. Synthetic ProTx-I was also made and exhibits the same potency as the native peptide. Synthetic ProTx-I, but not ProTx-II, also inhibits K(V) 2.1 channels with 10-fold less potency than its potency on Na(V) channels. These peptides represent novel tools for exploring the gating mechanisms of several Na(V) and Ca(V) channels.

The rat thyroid Na ؉ /I ؊ symporter (NIS) was expressed in Xenopus 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 Transporter density in the plasma membrane was determined using freeze-fracture electron microscopy. Expression of NIS in oocytes led to a ϳ2.5-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 Ϫ ) by the thyroid gland epithelium, previously referred to as the "iodide pump" or "iodide trap," is a Na ϩ -dependent secondary active transport process mediated by the Na1 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 Ϫ in the biosynthesis of thyroid hormones triiodothyronine and tetraiodothyronine, and to the significance of NIS in the diagnosis and treatment of thyroid disorders (1). A cDNA clone encoding NIS has recently been isolated, sequenced, and expressed in Xenopus laevis oocytes (2). Oocytes injected with NIS cRNA exhibit a 700-fold increase in perchlorate-sensitive I Ϫ uptake. This study reports a comprehensive characterization of rat NIS function expressed in X. laevis oocytes. NIS activity is Na ϩ -dependent and electrogenic, and the stoichiometry of cotransport is 2 Na ϩ :1 anion. Kinetics of transport as a function of external Na ϩ and substrate concentration suggest an ordered binding of Na ϩ and substrate to the transporter in which binding of Na ϩ 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 substrateuncoupled Na ϩ -dependent leak currents of NIS have provided insight into the nature of Na ϩ /I Ϫ cotransport. Combined data from electrophysiological measurements and freeze fracture electron microscopy suggest that NIS may be multimeric in its functional form. EXPERIMENTAL PROCEDURESNIS cRNA (50 ng) was microinjected into stage V-VI X. laevis oocytes (2, 3), 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 CaCl 2 , 1 MgCl 2 , 10 HEPES, pH 7.5. Chloride was replaced with gluconate in Cl Ϫ -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 wit...

The Na ؉ ͞I ؊ symporter (NIS) is the plasma membrane protein that catalyzes active I ؊ transport in the thyroid, the first step in thyroid hormone biogenesis. The cDNA encoding NIS was recently cloned in our laboratory and a secondary structure model proposed, suggesting that NIS is an intrinsic membrane protein (618 amino acids; Ϸ65.2 kDa predicted molecular mass) with 12 putative transmembrane domains. Here we report the generation of a site-directed polyclonal anti-COOH terminus NIS antibody (Ab) that immunoreacts with a Ϸ87 kDa-polypeptide present in membrane fractions from a rat thyroid cell line (FRTL-5). The modelpredicted cytosolic-side location of the COOH terminus was confirmed by indirect immunof luorescence experiments using anti-COOH terminus NIS Ab in permeabilized FRTL-5 cells. Immunoreactivity was competitively blocked by the presence of excess synthetic peptide. Treatment of membrane fractions from FRTL-5 cells, Xenopus laevis oocytes, and COS cells expressing NIS with peptidyl N-glycanase F converted the Ϸ87 kDa-polypeptide into a Ϸ50 kDa-species, the same relative molecular weight exhibited by NIS expressed in E. coli. Anti-NIS Ab immunoprecipitated both the NIS precursor molecule (Ϸ56 kDa) and the mature Ϸ87 kDa form. Furthermore, a direct correlation between circulating levels of thyroidstimulating hormone and NIS expression in vivo was demonstrated.

Insulin has a narrow therapeutic index, reflected in a small margin between a dose that achieves good glycemic control and one that causes hypoglycemia. Once injected, the clearance of exogenous insulin is invariant regardless of blood glucose, aggravating the potential to cause hypoglycemia. We sought to create a "smart" insulin, one that can alter insulin clearance and hence insulin action in response to blood glucose, mitigating risk for hypoglycemia. The approach added saccharide units to insulin to create insulin analogs with affinity for both the insulin receptor (IR) and mannose receptor C-type 1 (MR), which functions to clear endogenous mannosylated proteins, a principle used to endow insulin analogs with glucose responsivity. Iteration of these efforts culminated in the discovery of MK-2640, and its in vitro and in vivo preclinical properties are detailed in this report. In glucose clamp experiments conducted in healthy dogs, as plasma glucose was lowered stepwise from 280 mg/dL to 80 mg/dL, progressively more MK-2640 was cleared via MR, reducing by ∼30% its availability for binding to the IR. In dose escalations studies in diabetic minipigs, a higher therapeutic index for MK-2640 (threefold) was observed versus regular insulin (1.3-fold).