title: Molecular cloning and expression of a GABA receptor subunit from the crayfish Procambarus clarkii.
abstract: Molecular cloning has introduced an unexpected, large diversity of neurotransmitter hetero- oligomeric receptors. Extensive research on the molecular structure of the γ-aminobutyric acid receptor (GABAR) has been of great significance for understanding how the nervous system works in both vertebrates and invertebrates. However, only two examples of functional homo-oligomeric GABA-activated Cl(-) channels have been reported. In the vertebrate retina, the GABAρ1 subunit of various species forms homo-oligomeric receptors; in invertebrates, a cDNA encoding a functional GABA-activated Cl(-) channel has been isolated from a Drosophila melanogaster head cDNA library. When expressed in Xenopus laevis oocytes, these subunits function efficiently as a homo-oligomeric complex. To investigate the structure-function of GABA channels from the crayfish Procambarus clarkii, we cloned a subunit and expressed it in human embryonic kidney cells. Electrophysiological recordings show that this subunit forms a homo-oligomeric ionotropic GABAR that gates a bicuculline-insensitive Cl(-) current. The order of potency of the agonists was GABA > trans-4-amino-crotonic acid = cis-4-aminocrotonic acid > muscimol. These data support the notion that X-organ sinus gland neurons express at least two GABA subunits responsible for the formation of hetero-oligomeric and homo-oligomeric receptors. In addition, by in situ hybridization studies we demonstrate that most X-organ neurons from crayfish eyestalk express the isolated pcGABAA β subunit. This study increases the knowledge of the genetics of the crayfish, furthers the understanding of this important neurotransmitter receptor family, and provides insight into the evolution of these genes among vertebrates and invertebrates.
authors.affiliation: Departamento de Fisiología, Biofísica, y Neurociencias, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, México Distrito Federal, México.
authors.forename: Eric N
authors.initials: EN
authors.lastname: Jiménez-Vázquez
authors.affiliation: Programa de Neurociencias, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, Tlalnepantla de Baz, Estado de México, México.
authors.forename: Clara E
authors.initials: CE
authors.lastname: Díaz-Velásquez
authors.affiliation: Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, México.
authors.forename: R M
authors.initials: RM
authors.lastname: Uribe
authors.affiliation: Programa de Neurociencias, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, Tlalnepantla de Baz, Estado de México, México.
authors.forename: Juan M
authors.initials: JM
authors.lastname: Arias
authors.affiliation: Departamento de Fisiología, Biofísica, y Neurociencias, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, México Distrito Federal, México.
authors.forename: Ubaldo
authors.initials: U
authors.lastname: García
mesh_terms: D000818:Animals; D003400:Astacoidea / Q000235:genetics / Q000378:metabolism; D001703:Biophysics; D003001:Cloning, Molecular; D004558:Electric Stimulation; D018682:GABA Agents / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics; D008958:Models, Molecular; D018408:Patch-Clamp Techniques; D010802:Phylogeny; D021122:Protein Subunits / Q000235:genetics* / Q000378:metabolism*; D018079:Receptors, GABA / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection
chemical_list: D018682:GABA Agents; D021122:Protein Subunits; D018079:Receptors, GABA
keywords: X-organ neurons; crustacean; cys-loop family; homomeric ionotropic receptors; ligand-gated ion channel; β subunit