RESUMEN
This study reports the comparative molecular modeling, docking and dynamic simulations of human alpha9alpha10 nicotinic acetylcholine receptors complexed with acetylcholine, nicotine and alpha-conotoxin RgIA, using as templates the crystal structures of Aplysia californica and Lymnaea stagnalis acetylcholine binding proteins. The molecular dynamics simulations showed that Arg112 in the complementary alpha10(-) subunit, is a determinant for recognition in the site that binds small ligands. However, Glu195 in the principal alpha9(+), and Asp114 in the complementary alpha10(-) subunit, might confer the potency and selectivity to alpha-conotoxin RgIA when interacting with Arg7 and Arg9 of this ligand.
Asunto(s)
Modelos Moleculares , Receptores Nicotínicos/química , Acetilcolina/química , Aminoácidos , Animales , Aplysia/química , Sitios de Unión , Simulación por Computador , Conotoxinas/química , Humanos , Lymnaea/química , Nicotina/química , Unión ProteicaRESUMEN
A cDNA encoding a G-protein-coupled receptor was cloned from the central nervous system of the pond snail Lymnaea stagnalis. The predicted amino acid sequence of this cDNA most closely resembles the Drosophila tyramine/octopamine receptor, the Locusta tyramine receptor, and an octopamine receptor (Lym oa1) that we recently cloned from Lymnaea. After stable expression of the cDNA in HEK293 cells, we found that [3H]rauwolscine binds with high affinity to the receptor (KD = 6.2.10(-9) M). Octopamine appears to be the most potent naturally occurring agonist to displace the [3H]rauwolscine binding (Ki = 3.0.10(-7) M). Therefore, the receptor is considered to be an octopamine receptor and is consequently designated Lym oa2. The novel receptor shares little pharmacological resemblance with Lym oa1, indicating that the two receptors represent different octopamine receptor subfamilies. Octopaminergic stimulation of Lym oa2 does not induce changes in intracellular concentrations of cAMP or inositol phosphates. However, electrophysiological experiments indicate that octopamine is able to activate a voltage-independent Cl- current in HEK293 cells stably expressing Lym oa2. Although opening of this chloride channel most probably does not require the activation of either protein kinase A or C, it can be blocked by inhibition of protein phosphorylation.
Asunto(s)
Canales de Cloruro/metabolismo , Lymnaea/genética , Octopamina/fisiología , Receptores de Amina Biogénica/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Cultivadas , Clonación Molecular , ADN Complementario/genética , Proteínas de Unión al GTP/fisiología , Humanos , Activación del Canal Iónico , Lymnaea/química , Glicoproteínas de Membrana/genética , Datos de Secuencia Molecular , Técnicas de Placa-Clamp , Fosforilación , Transducción de Señal , Yohimbina/metabolismoRESUMEN
A full-length complementary DNA has been isolated, from the fresh-water mollusc Lymnaea stagnalis, that encodes a polypeptide (which we have named zeta) that exhibits between 30% and 40% identity to vertebrate GABAA and glycine receptor subunit sequences. The locations of seven introns have been determined in the corresponding gene and six of these occur at similar relative positions as those found in vertebrate GABAA receptor genes. RNase protection studies have revealed that the transcript for this Lymnaean polypeptide is present at highest levels in the adult nervous system but that it can also be detected in peripheral tissues.