Florence Kotzyba-Hibert

A photoaffinity ligand of the acetylcholine-binding site predominantly labels the region 179–207 of the α-subunit on native acetylcholine receptor from Torpedo marmorata

Regions of the Torpedo marmorata acetylcholine receptor (AChR) α‐subunit involved in the binding of acetylcholine were probed with two different covalent ligands. The sulfhydryl‐directed affinity reagent 4‐(N‐maleimido)phenyltrimethylammonium iodide labeled a single α‐subunit cyanogen bromide fragment on the reduced AChR which was identified as α 179–207. The novel photoaffinity ligand p‐(N,N‐dimethylamino)‐benzenediazonium fluoroborate, on the other hand, labeled three distinct α‐chain cyanogen bromide fragments on the unmodified AChR in a carbamylcholine‐protectable manner. The major radiolabeled species was purified and identified by sequence analysis as α 179–207. The acetylcholine‐binding site on the native AChR may thus involve several distinct portions of the α‐chain, with the region α 179–207 making a major contribution to the site.

Fluorescent Agonists for the Torpedo Nicotinic Acetylcholine Receptor

We have synthesized a series of fluorescent acylcholine derivatives carrying different linkers that vary in length and structure and connect the acylcholine unit to the environment‐sensitive fluorophores 7‐(diethylamino)coumarin‐3‐carbonyl (DEAC) or N‐(7‐nitrobenz‐2‐oxa‐1,3‐diazol‐yl) (NBD). The pharmacological properties of the fluorescent analogues were investigated on heterologously expressed nicotinic acetylcholine receptor (nAChR) from Torpedo californica and on oocytes transplanted with nAChR‐rich Torpedo marmorata membranes. Agonist action strongly depends on the length and the structure of the linker. One particular analogue, DEAC‐Gly‐C6‐choline, showed partial agonist behavior with about half of the maximum response of acetylcholine, which is at least 20 times higher than those observed with previously described fluorescent dansyl‐ and NBD‐acylcholine analogues. Binding of DEAC‐Gly‐C6‐choline to Torpedo nAChR induces a strong enhancement of fluorescence intensity. Association and displacement kinetic experiments revealed dissociation constants of 0.5 nM for the αδ‐binding site and 15.0 nM for the αγ‐binding site. Both the pharmacological and the spectroscopic properties of this agonist show great promise for characterizing the allosteric mechanism behind the function of the Torpedo nAChR, as well as for drug‐screening studies.

Dynamic Structural Investigations on the Torpedo Nicotinic Acetylcholine Receptor by Time-Resolved Photoaffinity Labeling

An increasing number of high‐resolution structures of membrane‐embedded ion channels (or soluble homologues) have emerged during the last couple of years. The most pressing need now is to understand the complex mechanism underlying ion‐channel function. Time‐resolved photoaffinity labeling is a suitable tool for investigating the molecular function of membrane proteins, especially when high‐resolution structures of related proteins are available. However until now this methodology has only been used on the Torpedo nicotinic acetylcholine receptor (nAChR). nAChRs are allosteric cation‐selective receptor channels that are activated by the neurotransmitter acetylcholine (ACh) and implicated in numerous physiological and pathological processes. Time‐resolved photoaffinity labeling has already enabled local motions of nAChR subdomains (i.e. agonist binding sites, ion channel, subunit interface) to be understood at the molecular level, and has helped to explain how small molecules can exert their physiological effect, an important step toward the development of drug design. Recent analytical and technical improvements should allow the application of this powerful methodology to other membrane proteins in the near future.

Aryldiazonium salts as photoaffinity labels of the nicotinic acetylcholine receptor PCP binding site

Several aryldiazonium salts are described as irreversible blockers of the phencyclidine binding site of the nicotinic cholinergic receptor. A partial hydrophobic character increases the affinity of these salts for the phencyclidine binding site. Photoaffinity labelling with a tritiated diazonium salt in the presence of either carbamylcholine or α‐bungarotoxin leads to incorporation of radioactivity into the 4 subunits of the receptor. Among these diazonium salts, an imidazole derivative is unique in that the photoinduced irreversible blocking in only effective when the receptor is in a desensitised state.

Structural Reorganization of the Acetylcholine Binding Site of the Torpedo Nicotinic Receptor as Revealed by Dynamic Photoaffinity Labeling

We explored the structural changes that occur at the acetylcholine binding site of the Torpedo marmorata nicotinic receptor during activation by the tritiated photoactivatable agonist (diazocyclohexadienoylpropyl)trimethylammonium ([3H]DCTA). We quantified the incorporation of radioactivity into the receptor subunits as a function of the mixing time of [3H]DCTA with the receptor by using a rapid‐mixing device adapted with a photochemical quenching system. A saturable increase of the specific photolabeling on the α and γ subunits was observed with a half‐time of about 2 minutes. We further analyzed this photoincorporation either after rapid mixing for 500 ms or after equilibration for 50 minutes. Under these conditions, [3H]DCTA explored transient state(s) and the stable desensitized state, respectively. Comparative analyses showed that at a probe concentration of 10 μM the relative variation of photoincorporation was more pronounced for the γ subunit (three‐ to fourfold) than for the α subunit (about twofold). By contrast, the relative distribution of radioactivity among α‐subunit labeled residues (αTyr190, αCys192, αCysC193, and αTyr198) did not change. Altogether, these results reveal that during the course of agonist‐induced receptor desensitization, the site‐lining peptide loops, which belong to adjacent α and γ subunits, move closer to each other.

Photochemical labeling of membrane-associated and channel-forming domains of proteins directed by energy transfer

Singlet—singlet energy transfer reactions from excited tryptophan residues to photoactivatable probes possessing a suitable chromophore, generate reactive species in the vicinity of the protein, leading to its covalent labeling. This delayed labeling process can be used to map the membrane‐surrounded regions of proteins with improved efficiency when it is applied with appropriate photoactivatable phospholipids. The same principle could also be applied to the labeling of channel‐forming transmembrane domains of ion channels, provided that suitable photoactivatable permeant ions were available. Both applications will be discussed with regard to their potential and feasibility.

Molecular investigations on the nicotinic acetylcholine receptor: conformational mapping and dynamic exploration using photoaffinity labeling.

The nicotinic acetylcholine receptor (nAChR) is a well-understood member of the ligand-gated ion channels superfamily. The members of this signaling proteins group, including 5HT3, GABAA, glycine, and ionotropic glutamate receptors, are thought to share common secondary, tertiary, and quaternary structures on the basis of a very high degree of sequence similarity. Despite the absence of X-ray crystallographic data, considerable progress on structural analysis of nAChR was achieved from biochemical, mutational, and electron microscopy data allowing the emergence of a three-dimensional image. Photoaffinity labeling and site-directed mutagenesis gave information on the tertiary structure with respect to the agonist/antagonist binding sites, the ion channel, and its selectivity filter.nAChR is an allosterical protein that undergoes interconversion among several conformational states. Time-resolved photolabeling was used in an attempt to elucidate the structural changes that occur in nAChR on neurotransmitter activation. Tertiary and quaternary rearrangements were found in the cholinergic binding pocket and in the channel lumen, but the structural determinant and the functional link between the binding of agonist and the channel gating remain unknown. Time-resolved photolabeling of the functional activated A state using photosensitive agonists might help in understanding the dynamic process leading to the interconversion of the different states.

Syntheses and biological properties of cysteine-reactive epibatidine derivatives.

The synthesis of epibatidine derivatives modified at the 2-position of the pyridine or pyrimidine rings by reactive functions are described for potential irreversible site-directed coupling reactions on cysteine mutants of neuronal nicotinic acetylcholine receptors. An improved synthesis of the 7-azabicyclo[2,2,1]hepta-2,5-diene key intermediate has been developed to allow reproducible syntheses of the epibatidine derivatives. Binding tests and electrophysiological experiments allowed to select the 2-substituted alpha-chloroacetamido 13 and the chloropyrimidine derivative 11 as potential site-directed probes for the epibatidine binding site.

Novel Photoactivatable Agonist of the Nicotinic Acetylcholine Receptor of Potential Use for Exploring the Functional Activated State

Abstract: The nicotinic acetylcholine receptor (AChR) exhibits at least four different conformational states varying in affinity for agonists such as acetylcholine (ACh). Photoaffinity labeling has been previously used to elucidate the topography of the AChR. However, to date, the photosensitive probes used to explore the cholinergic binding site photolabeled only closed or desensitized states of the receptor. To identify the structural modifications occurring at the ACh binding site on allosteric transition associated with receptor activation, we have investigated novel photoactivatable 4‐diazocyclohexa‐2,5‐dienone derivatives as putative cholinergic agonists. Such compounds are fairly stable in the dark and generate highly reactive carbenic species on irradiation. In binding experiments using AChRs from Torpedo marmorata, these ligands had affinities for the ACh binding site in the micromolar range and did not interact with the noncompetitive blocker site (greater than millimolar affinity). Irreversible photoinactivation of ACh binding sites was obtained with the ligand 1b (up to 42% at 500 µM) in a protectable manner. In patch‐clamp studies, 1b was shown to be a functional agonist of peripheral AChR in TE 671 cells, with the interesting property of exhibiting no or very little desensitization even at high concentrations.

NICOTINIC ACETYLCHOLINE RECEPTOR LABELED WITH A TRITIATED, PHOTOACTIVATABLE AGONIST : A NEW TOOL FOR INVESTIGATING THE FUNCTIONAL, ACTIVATED STATE

Upon agonist activation, the nicotinic acetylcholine receptor undergoes allosteric transitions leading to channel opening and sodium ion influx. The molecular structure of the agonist binding site has been mapped previously by photoaffinity labeling, but most photosensitive probes used for this purpose interact only with closed receptor states (resting or desensitized). We have synthesized two novel photoactivatable 4-diazocyclohexa-2,5-dienone derivatives as cholinergic agonist candidates, with the objective of identifying structural changes at the acetylcholine binding site associated with receptor activation. One of these ligands, 9b, is a functional agonist at muscle acetylcholine receptors in human TE 671 cells. In photolabeling experiments with 9b, up to 35% inactivation of agonist binding sites was observed at Torpedo acetylcholine receptors. Tritiated 9b was synthesized, and photolabeling was found to occur mainly on the alpha-subunit in a partially protectable manner. This novel radiolabeled photoprobe appears to be suitable for future investigation of the molecular dynamics of allosteric transitions occurring at the active acetylcholine receptor binding site.