Enz R, Cutting G R
Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA.
Eur J Neurosci. 1999 Jan;11(1):41-50. doi: 10.1046/j.1460-9568.1999.00423.x.
In the central nervous system, receptors for gamma-aminobutyric acid (GABA) are responsible for inhibitory neurotransmission. Anatomical and electrophysiological studies indicate that GABAC receptors are composed of rho subunits. While the rho 1 subunit of various species forms homooligomeric receptors with GABAC-like properties, molecular cloning has identified additional rho subunits whose functional role is unclear. By RT-PCR, we demonstrated that rho 1 expression is primarily restricted to the retina, whereas the rho 2 subunit was present in all brain regions tested. Transfection of HEK-293 cells with rho 2 cDNA resulted in GABA-gated whole-cell currents that differed from those mediated by the rho 1 subunit in two respects: maximal amplitude (rho 1:rho 2 approximately 4:1) and inactivation time course (rho 1:rho 2 approximately 2:1). Cotransfection of rho 1 and rho 2 cDNA in a 1:1 ratio generated whole-cell currents with large amplitudes characteristic of rho 1 but more rapid inactivation typical for rho 2. This observation suggested formation of heterooligomeric GABAC receptors with distinct features. Therefore, we tested the assembly of rho 1 and rho 2 subunits by cotransfecting rho 2 cDNA together with a chimeric rho 1 beta 1 subunit, known to interfere with rho 1 assembly in a dominant-negative fashion. Reduction of rho 2 generated currents correlated with the ratio of chimeric to rho 2 cDNA. Secondly, we determined that the picrotoxinin sensitivity of cells transfected with various ratios of rho 1 and rho 2 cDNA differed from that expected of a pure mixture of homooligomeric receptors. The latter two observations support the idea that rho 1 and rho 2 subunits form heterooligomeric GABAC receptors in mammalian cells. Together, our results indicate that the presence of both rho subunits enables the formation of heterooligomeric receptors with physical properties distinct from homooligomers, thus increasing the diversity of GABAC receptors in the CNS.
在中枢神经系统中，γ-氨基丁酸（GABA）受体负责抑制性神经传递。解剖学和电生理学研究表明，GABAC受体由rho亚基组成。虽然不同物种的rho 1亚基形成具有GABAC样特性的同聚体受体，但分子克隆已鉴定出其他rho亚基，其功能作用尚不清楚。通过逆转录聚合酶链反应（RT-PCR），我们证明rho 1的表达主要局限于视网膜，而rho 2亚基存在于所有测试的脑区。用rho 2 cDNA转染人胚肾293（HEK-293）细胞产生了GABA门控全细胞电流，该电流在两个方面不同于由rho 1亚基介导的电流：最大幅度（rho 1:rho 2约为4:1）和失活时间进程（rho 1:rho 2约为2:1）。以1:1的比例共转染rho 1和rho 2 cDNA产生了具有rho 1特征性大振幅但具有rho 2典型更快失活的全细胞电流。这一观察结果提示形成了具有独特特征的异聚体GABAC受体。因此，我们通过将rho 2 cDNA与已知以显性负性方式干扰rho 1组装的嵌合rho 1β1亚基共转染来测试rho 1和rho 2亚基的组装。rho 2产生电流的减少与嵌合体与rho 2 cDNA的比例相关。其次，我们确定用不同比例的rho 1和rho 2 cDNA转染的细胞对印防己毒素（picrotoxinin）的敏感性不同于同聚体受体纯混合物的预期敏感性。后两个观察结果支持rho 1和rho 2亚基在哺乳动物细胞中形成异聚体GABAC受体的观点。总之，我们的结果表明，两种rho亚基的存在能够形成具有不同于同聚体物理性质的异聚体受体，从而增加了中枢神经系统中GABAC受体的多样性。
