Jiménez-Vázquez Eric N, Díaz-Velásquez Clara E, Uribe R M, Arias Juan M, García Ubaldo
Departamento de Fisiología, Biofísica, y Neurociencias, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, México Distrito Federal, México.
Programa de Neurociencias, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, Tlalnepantla de Baz, Estado de México, México.
J Neurosci Res. 2016 Feb;94(2):190-203. doi: 10.1002/jnr.23695. Epub 2015 Nov 17.
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.
分子克隆技术揭示了神经递质异源寡聚体受体存在意想不到的大量多样性。对γ-氨基丁酸受体（GABAR）分子结构的广泛研究，对于理解脊椎动物和无脊椎动物神经系统的工作方式具有重要意义。然而，仅报道了两个功能性同聚体GABA激活的Cl⁻通道的例子。在脊椎动物视网膜中，不同物种的GABAρ1亚基形成同聚体受体；在无脊椎动物中，已从黑腹果蝇头部cDNA文库中分离出编码功能性GABA激活的Cl⁻通道的cDNA。当在非洲爪蟾卵母细胞中表达时，这些亚基作为同聚体复合物高效发挥作用。为了研究克氏原螯虾GABA通道的结构与功能，我们克隆了一个亚基并在人胚肾细胞中进行表达。电生理记录表明，该亚基形成一种同聚体离子型GABAR，可控制对荷包牡丹碱不敏感的Cl⁻电流。激动剂的效力顺序为GABA > 反式-4-氨基巴豆酸 = 顺式-4-氨基巴豆酸 > 蝇蕈醇。这些数据支持了X器官-窦腺神经元表达至少两种负责形成异源寡聚体和同聚体受体的GABA亚基这一观点。此外，通过原位杂交研究，我们证明克氏原螯虾眼柄的大多数X器官神经元表达分离出的pcGABAA β亚基。这项研究增加了对克氏原螯虾遗传学的认识，进一步加深了对这个重要神经递质受体家族的理解，并为这些基因在脊椎动物和无脊椎动物中的进化提供了见解。
