RESUMEN
Torpedo marmorata acetylcholine binding sites were photolabeled using 360 nm light, at equilibrium in the desensitized state, with the agonist [3H]DCTA utilizing the CeIV/glutathione procedure described previously (Grutter, et al. (1999) Biochemistry 38, 7476-7484). Photoincorporation of [3H]DCTA was concentration-dependent with a maximum of 7.5% specific labeling on the alpha-subunit and 1.2% on the gamma-subunit. The apparent dissociation constants for labeling of the alpha- and gamma-subunits were 2.2 +/- 1.1 and 3.6 +/- 2.8 microM, respectively. The alpha-chains isolated from receptor-rich membranes photolabeled in the absence or in the presence of carbamylcholine were cleaved with CNBr using an efficient "in gel" procedure. The resulting peptide fragments were purified by HPLC and further submitted to trypsinolysis. The digest was analyzed by HPLC leading to a single radioactive peak which, by microsequencing, revealed two sequences extending from alpha Lys-179 and from alpha His-186, respectively. Radioactive signals could be unambiguously attributed to positions corresponding to residues alpha Tyr-190, alpha Cys-192, alpha Cys-193, and alpha Tyr-198. These four identified [3H]DCTA-labeled residues, which have been also labeled with other affinity and photoaffinity probes including the agonist [3H]nicotine, belong to loop C of the ACh binding site. The chemical structure of [3H]DCTA, together with its well-defined and powerful photochemical reactivity, provides convincing evidence that loop C-labeled residues are primarily involved in the interaction with the ester moiety of acetylcholine.
Asunto(s)
Acetilcolina/metabolismo , Aminoácidos/metabolismo , Compuestos de Diazonio/metabolismo , Agonistas Nicotínicos/metabolismo , Etiquetas de Fotoafinidad/metabolismo , Receptores Nicotínicos/metabolismo , Secuencia de Aminoácidos , Animales , Membrana Celular/metabolismo , Bromuro de Cianógeno , Ésteres , Hidrólisis , Datos de Secuencia Molecular , Fragmentos de Péptidos/metabolismo , Unión Proteica , Torpedo , Tritio , Tripsina/metabolismoRESUMEN
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, GABA(A), 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.
Asunto(s)
Conformación Proteica , Receptores Nicotínicos/química , Receptores Nicotínicos/fisiología , Animales , Canales Iónicos/fisiología , Sustancias Macromoleculares , Modelos Moleculares , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
The molecular structure of Torpedo marmorata acetylcholine binding sites has been investigated previously by photoaffinity labeling. However, besides the nicotine molecule [Middleton et al. (1991) Biochemistry 30, 6987-6997], all other photosensitive probes used for this purpose interacted only with closed receptor states. In the perspective of mapping the functional activated state, we synthesized and developed a new photoactivatable agonist of nAChR capable of alkylation of the acetylcholine (ACh) binding sites, as reported previously [Kotzyba-Hibert et al. (1997) Bioconjugate Chem. 8, 472-480]. Here, we describe the setup of experimental conditions that were made in order to optimize the photolabeling reaction and in particular its specificity. We found that subsequent addition of the oxidant ceric ion (CeIV) and reduced glutathione before the photolabeling step lowered considerably nonspecific labeling (over 90% protection with d-tubocurarine) without affecting the binding properties of the ACh binding sites. As a consequence, irradiation at 360 nm for 20 min in these new conditions gave satisfactory coupling yields (7.5%). A general mechanism was proposed to explain the successive reactions occurring and their drastic effect on the specificity of the labeling reaction. Last, these incubation conditions can be extended to nanosecond pulsed laser photolysis leading to the same specific photoincorporation as for usual irradiations (8.5% coupling yield of ACh binding sites, 77% protection with carbamylcholine). Laser flash photocoupling of a diazocyclohexadienoyl probe on nAChR was achieved for the first time. Taken together, these data indicate that future investigation of the molecular dynamics of allosteric transitions occurring at the activated ACh binding sites should be possible.
Asunto(s)
Cerio/metabolismo , Glutatión/metabolismo , Agonistas Nicotínicos/metabolismo , Etiquetas de Fotoafinidad/metabolismo , Receptores Nicotínicos/metabolismo , Animales , Sitios de Unión , Cerio/química , Cromatografía Líquida de Alta Presión , Compuestos de Diazonio/química , Glutatión/química , Hidroquinonas/metabolismo , Rayos Láser , Ligandos , Agonistas Nicotínicos/química , Oxidantes/química , Oxidantes/metabolismo , Etiquetas de Fotoafinidad/química , Fotólisis , Quinonas/metabolismo , Receptores Nicotínicos/química , Torpedo , TritioRESUMEN
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.
Asunto(s)
Receptores Nicotínicos/química , Animales , Línea Celular , Humanos , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Sondas Moleculares , Agonistas Nicotínicos/farmacología , Técnicas de Placa-Clamp , Fotoquímica , Receptores Nicotínicos/efectos de los fármacos , Receptores Nicotínicos/metabolismo , Torpedo , TritioRESUMEN
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 microM) 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.
Asunto(s)
Marcadores de Afinidad/química , Compuestos de Diazonio/química , Agonistas Nicotínicos/química , Agonistas Nicotínicos/farmacología , Fotoquímica , Acetilcolina/farmacología , Animales , Sitios de Unión/fisiología , Electrofisiología , Ligandos , Agonistas Nicotínicos/metabolismo , Antagonistas Nicotínicos/química , Antagonistas Nicotínicos/metabolismo , Antagonistas Nicotínicos/farmacología , TorpedoRESUMEN
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.
Asunto(s)
Marcadores de Afinidad , Transferencia de Energía , Canales Iónicos/metabolismo , Proteínas de la Membrana/metabolismo , Membrana Celular/metabolismo , Canales Iónicos/química , Proteínas de la Membrana/química , Fosfolípidos/metabolismo , Fotoquímica , Triptófano/metabolismoRESUMEN
The regioselective modification of a snake curaremimetic toxin is described. Toxin-alpha from Naja nigricollis was derivatized with an electron-dense maleimido undecagold cluster 5. The cluster-bound obtained toxin 8 has a very high affinity for the cholinergic binding site (Ki = 70 pM) of Torpedo marmorata nicotinic receptor. It is harboring a compact heavy atom core of about 8 A which makes it very useful for electron microscopy experiments.
Asunto(s)
Proteínas Neurotóxicas de Elápidos/síntesis química , Antagonistas Nicotínicos , Animales , Sitios de Unión , Proteínas Neurotóxicas de Elápidos/química , Proteínas Neurotóxicas de Elápidos/metabolismo , Técnicas In Vitro , Estructura Molecular , Compuestos Orgánicos de Oro , Compuestos Organometálicos/síntesis química , Compuestos Organometálicos/química , Compuestos Organometálicos/metabolismo , Receptores Nicotínicos/metabolismo , Torpedo/metabolismoRESUMEN
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.
Asunto(s)
Acetilcolina/metabolismo , Compuestos de Diazonio/metabolismo , Receptores Colinérgicos/metabolismo , Marcadores de Afinidad/metabolismo , Aminoácidos/análisis , Animales , Membrana Celular/metabolismo , Bromuro de Cianógeno , Órgano Eléctrico/metabolismo , Sustancias Macromoleculares , Fragmentos de Péptidos/análisis , Receptores Colinérgicos/aislamiento & purificación , TorpedoRESUMEN
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 alpha-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.