Binding sites of Torpedo acetylcholinesterase (EC 3.1.1.7) for quaternary ligands were investigated by x-ray crystallography and photoaffinity labeling. Crystal structures ofcomplexes with ligands were determined at 2.8-A resolution. In a complex with edrophonium, the quaternary nitrogen of the ligand interacts with the indole of Trp-84, and Its m-hydroxyl displays bilbrcated hydrogen bonding to two members of the catalytic triad, Ser-200 and Hls-440. In a complex with tacrine, the acridine Is stacked against the indole of Trp-84. The bisquaternary ligand decamethonium Is oriented along the narrow gorge leading to the active site; one quaternary group Is apposed to the indole of Trp-84 and the other to that of Trp-279, near the top of the gorge. The only major conformational difference between the three complexes is in the orientation of the phenyl ring of Phe-330. In the decamethonium complex it lies parallel to the surface of the gorge; in the other two complexes it is positioned to make contact with the bound ligand. This dose Interaction was confirmed by photoaffnity labeling by the photosensitive probe 3H-labeled p-(N,Ndimethylamlno)benzenediazonium fluoroborate, which labeled, predominantly, Phe-330 within the active dte. Labeling ofTrp-279 was also observed. One mole oflabel is incorporated per mole of AcChoEase inactivated, indicating that labeling of Trp-279 and that of Phe-330 are mutually exclusive. The structural and chemical data, together, show the important role ofaromatic groups as binding sites for quaternary ligands, and they provide complementary evidence assigning and Phe-330 to the "anionic" subsite of the active site and Trp-279 to the "peripheral" anionic site.

A random primed expression cDNA library was constructed from the RNA of NG 108-15 cells. Pools of plasmid DNA were transfected into COS cells, which were screened for their ability to bind 'H-labeled Tyr-D-Thr-GlyPhe-Leu-Thr, a tritiated agonist for the -opioid receptor. A cDNA was isolated that encodes a 371-amino acid-residue protein presenting all the structural characteristics of receptors that interact with guanine nucleotide-binding proteins. Opioid receptors have long been described as membrane receptors of the nervous system mediating the analgesic effects of opium-derived alkaloids. Endogeneous ligands and their precursors have been characterized, and their role in response to pain and stress has been widely studied (1). Pharmacological studies have shown the existence of three subtypes of receptors, ,u (morphine), 8 (enkephalin), and K (dynorphin), the cellular inhibitory actions of which are linked to G protein activation (2). Opioid receptors are, therefore, believed to be part of the G protein-coupled receptor (GPR) family, a class of membrane-bound receptors that exhibit a seven-transmembrane-spanning domain topology and represent 80% of all known receptors (3).Several attempts to clone cDNAs encoding opioid receptors have been reported. A cDNA encoding an opioid-binding protein (OBCAM) with g selectivity was isolated (4), but the predicted protein lacks transmembrane domains, presumed necessary for signal transduction. More recently, the isolation of another cDNA was reported, which was obtained by expression cloning (5). The deduced protein sequence displays seven putative transmembrane domains and is very similar to the human neuromedin K receptor. However, the affinity of opioid ligands for this receptor expressed in COS cells is two orders of magnitude below the expected value, and no subtype selectivity can be shown. Finally, attempts to clone opioid receptors via approaches relying on sequence similarities in the GPR family, using the PCR technology, were unsuccessful. There is, therefore, no convincing evidence that a cDNA encoding an opioid receptor has been cloned.We report here the isolation of a cDNA encoding a 8-opioid receptorA We used a transient expression strategy where a cDNA library derived from NG 108-15 cells was screened using 3H-labeled Tyr-D-Thr-Gly-Phe-Leu-Thr (DTLET), a tritiated agonist for the 8-opioid receptor. Our pharmacological study of the cloned cDNA expressed in COS cells shows that it possesses all expected properties of a 8 receptor. A comparative analysis of the deduced protein sequence with other members of the GPR family is also presented.MATERIALS AND METHODS Library Construction and Screening. NG 108-15 cells were provided by B. Foucaud (URA 1836, Facultd de Pharmacie, Strasbourg, France). Cells were harvested at 50% confluency, RNA was prepared by LiCl/urea precipitation (6), and polyadenylylated RNA was purified with an oligo(dT) column (Pharmacia). The cDNA library construction in the mammalian expression vector pCDM8 has been described (7). Plas...

In 175 patients with chronic obstructive lung disease (157 chronic bronchitic and 18 emphysematous patients) exhibiting moderate to severe airway obstruction (mean FEV1/vital capacity = 40X2 + 11 1 %), cumulative survival rates calculated by the actuarial method were compared in subgroups according to the initial level of mean pulmonary artery pressure, pulmonary volumes, and arterial blood gases. Patients were catheterised between 1968 and 1972 and were followed for at least five years. The results emphasise the high prognostic value of PAP since survival rates after four and seven years were significantly lower in the subgroup with PAP > 20 mmHg (2.7 kPa).Certain other parameters ("driving" pressure across_the pulmonary circulation, FEV, and PaCo2) appear to be equally good at predicting survival as PAP in these obstructed patients. The effect of age should be taken into account in prognostic studies such as ours since survival rates were significantly lower in patients over 60 years of age. In 64 patients who underwent a second right heart catheterisation at least three years after the first (average delay: 5*5 + 2 years), the prognostic value of changes in PAP, arterial blood gases, and pulmonary volumes was studied but with the exception of Pao2 was unremarkable. Further studies are needed in this field.In chronic obstructive pulmonary disease (COPD) and especially in chronic bronchitis, the presence of cor pulmonale is associated with a poor prognosis.1-3 However pulmonary arterial hypertension is measurable and therefore easier to assess than cor pulmonale and prognostic studies have usually been related to the presence of pulmonary hypertension and its severity.4-7 Most of these studies have included cases of severe COPD with a high proportion of patients having experienced episodes of rightsided heart failure. Only one recent study7 was devoted to the prognosis of pulmonary hypertension in a large series of patients with mild or moderate airflow obstruction. Follow-up data were generally not taken into account.We have investigated the prognostic value of mean pulmonary artery pressure (PAP) taking into account the influence of age, and compared it with other functional data (FEV1, arterial blood gases) in our series of patients with moderate to severe airway obstruction. The actuarial method was used to assess survival rate. In 64 patients who underwent a second

The acetylcholine-binding sites on the native, membrane-bound acetylcholine receptor from Torpedo marmorata were covalently labeled with the photoaffinity reagent [3H]-p-(dimethylamino)-benzenediazonium fluoroborate (DDF) in the presence of phencyclidine by employing an energy-transfer photolysis procedure. The alpha-chains isolated from receptor-rich membranes photolabeled in the absence or presence of carbamoylcholine were cleaved with CNBr and the radiolabeled fragments purified by high-performance liquid chromatography. Amino acid and/or sequence analysis demonstrated that the alpha-chain residues Trp-149, Tyr-190, Cys-192, and Cys-193 and an unidentified residue(s) in the segment alpha 31-105 were all labeled by the photoaffinity reagent in an agonist-protectable manner. The labeled amino acids are located within three distinct regions of the large amino-terminal hydrophilic domain of the alpha-subunit primary structure and plausibly lie in proximity to one another at the level of the acetylcholine-binding sites in the native receptor. These findings are in accord with models proposed for the transmembrane topology of the alpha-chain that assign the amino-terminal segment alpha 1-210 to the synaptic cleft. Furthermore, the results suggest that the four identified [3H]DDF-labeled residues, which are conserved in muscle and neuronal alpha-chains but not in the other subunits, may be directly involved in agonist binding.

Structural changes occurring upon desensiti- were labeled in a roughly identical manner in both resting and desensitized conformations, the labeling of Tyr-93 and Trp-149 increased up to 6-fold in the desensitized state. Tyr-93 and Trp-149 belong to separate regions containing strictly conserved "canonical" amino acids, common to all nicotinic, y-aminobutyrate, and glycine receptor subunits. These regions are thus likely to play a critical role in the regulation of ligand-gated ion channels.Recombinant DNA technology has led to the identification of a large collection of amino acid sequences from several members of the superfamily of ligand-gated ion channels, such as the nicotinic acetylcholine receptor (AcChoR) from electric organ, muscle, and brain, as well as the glycine, 'y-aminobutyrate (GABA), and glutamate receptors (reviewed in refs. 1-15). Structural and functional evidence supports the view that these allosteric membrane proteins (4, 5) are heteropentameric oligomers made up of at least two different categories of subunits, their distinctive pharmacological and physiological properties being associated with a defined subunit composition (6-9, 37, 38). Yet, in the absence of x-ray crystallographic data, little is known about the actual three-dimensional architecture of these oligomers and about the detailed structural mechanisms of the allosteric transitions that mediate fast and slow regulation of ion channel opening.One of the best characterized members of this family is the peripheral nicotinic AcChoR receptor from Torpedo electric organ (4,5,(10)(11)(12). It is an a2,83y oligomer of 300 kDa that contains two acetylcholine (AcCho) binding sites with distinct pharmacological properties and several allosteric sites for pharmacological agents referred to as noncompetitive blockers (4,5). New insights about the tertiary structure and amino acid composition of its binding sites for cholinergic ligands were recently obtained with the photoaffinity ligand p-(N,Ndimethyl)aminobenzenediazonium fluoroborate (DDF) (13)(14)(15). This compound, which behaves in the dark as a reversible competitive antagonist, covalently attaches to the cholinergic binding sites with a 1:1 stoichiometry upon photoactivation by energy transfer from the protein (13). The amino acids labeled by DDF belong to three distinct peptide segments of the large amino-terminal hydrophilic domain of the a subunit, which include Tyr-93, 15), and the two cysteines, 192 and 193, initially identified (16) with a maleimido derivative. Furthermore, DDF also reacts, though to a smaller extent, with the ,B, y, and 8 subunits (13), suggesting that they may each participate in one of the two binding areas in conjunction with one a subunit (17-20) and thereby account for the distinct binding specificity of the two a subunits (21,22).The present work aims at the analysis, at the amino acid level, of the structural reorganizations that occur in the cholinergic ligand-binding domains upon desensitization, a slow reaction (100 msec to 1 min) observ...