Department of Anesthesiology, Pharmacology and Therapeutics, University of British Columbia, Vancouver, British Columbia, Canada.
Neuroscience. 2012 Jan 10;201:85-95. doi: 10.1016/j.neuroscience.2011.10.049. Epub 2011 Nov 4.
The R-enantiomer of isovaline, an analgesic amino acid, has a chemical structure similar to glycine and GABA. Although its actions on thalamic neurons are strychnine-resistant and independent of the Cl(-) gradient, R-isovaline increases membrane conductance for K(+). The purpose of this study was to determine if R-isovaline activated metabotropic GABA(B) receptors. We used whole-cell voltage-clamp recordings to characterize the effects of R-isovaline applied by bath perfusion and local ejection from a micropipette to thalamic neurons in 250 μm thick slices of rat brain. The immunocytochemical methods that we employed to visualize GABA(B1) and GABA(B2) receptor subunits showed extensive staining for both subunits in ventrobasal nuclei, which were the recording sites. Bath or local application of R-isovaline caused a slowly developing increase in conductance and outward rectification in 70% (54/77) of neurons, both effects reversing near the K(+) Nernst potential. As with the GABA(B) agonist baclofen, G proteins likely mediated the R-isovaline effects because they were susceptible to blockade by non-hydrolyzable substrates of guanosine triphosphate. The GABA(B) antagonists CGP35348 and CGP52432 prevented the conductance increase induced by R-isovaline, applied by bath or local ejection. The GABA(B) allosteric modulator CGP7930 enhanced the R-isovaline induced increase in conductance. At high doses, antagonists of GABA(A), GABA(C), glycine(A), μ-opioid, and nicotinic receptors did not block R-isovaline responses. The observations establish that R-isovaline increases the conductance of K(+) channels coupled to metabotropic GABA(B) receptors. Remarkably, not all neurons that were responsive to baclofen responded to R-isovaline. The R-isovaline-induced currents outlasted the fast baclofen responses and persisted for a 1-2-h period. Despite some similar actions, R-isovaline and baclofen do not act at identical GABA(B) receptor sites. The binding of R-isovaline and baclofen to the GABA(B) receptor may not induce the same conformational changes in receptor proteins or components of the intracellular signaling pathways.
异亮氨酸的 R-对映体是一种镇痛氨基酸，其化学结构与甘氨酸和 GABA 相似。尽管其对丘脑神经元的作用不受士的宁影响且与 Cl(-)梯度无关，但 R-异亮氨酸增加了 K(+)的膜电导。本研究的目的是确定 R-异亮氨酸是否激活代谢型 GABA(B)受体。我们使用全细胞膜片钳记录技术，通过灌流和局部微管内注射的方式，在大鼠脑 250μm 厚切片的丘脑神经元上，对 R-异亮氨酸的作用进行了特征描述。我们采用免疫细胞化学方法来可视化 GABA(B1)和 GABA(B2)受体亚基，结果显示这两个亚基在腹侧基底核（记录部位）中均有广泛的染色。R-异亮氨酸的灌流或局部应用可引起 70%（54/77）的神经元的电导缓慢增加和外向整流，这两种作用均在接近 K(+)Nernst 电位时逆转。与 GABA(B)激动剂巴氯芬一样，G 蛋白可能介导了 R-异亮氨酸的作用，因为它们易受鸟嘌呤核苷酸三磷酸不可水解底物的阻断。GABA(B)拮抗剂 CGP35348 和 CGP52432 可阻止 R-异亮氨酸引起的由灌流或局部喷射引起的电导增加。GABA(B)变构调节剂 CGP7930 增强了 R-异亮氨酸诱导的电导增加。在高剂量下，GABA(A)、GABA(C)、甘氨酸(A)、μ-阿片、烟碱型受体的拮抗剂均不能阻断 R-异亮氨酸的反应。这些观察结果证实 R-异亮氨酸可增加与代谢型 GABA(B)受体偶联的 K(+)通道的电导。值得注意的是，并非所有对巴氯芬有反应的神经元都对 R-异亮氨酸有反应。R-异亮氨酸诱导的电流持续时间长于快速的巴氯芬反应，持续 1-2 小时。尽管有一些相似的作用，但 R-异亮氨酸和巴氯芬并不作用于相同的 GABA(B)受体位点。R-异亮氨酸和巴氯芬与 GABA(B)受体的结合可能不会引起受体蛋白或细胞内信号转导途径组成部分的相同构象变化。
