García-Alcocer G, García-Colunga J, Martínez-Torres A, Miledi R
Centro de Neurobiologia, Universidad Nacional Autónoma de México, Campus Juriquilla, Apartado Postal 1-1141, Juriquilla, Querétaro 76001, México.
Proc Natl Acad Sci U S A. 2001 Feb 27;98(5):2781-5. doi: 10.1073/pnas.031580798. Epub 2001 Feb 13.
A study was made of glycine (Gly) and gamma-aminobutyric acid (GABA) receptors expressed in Xenopus oocytes injected with rat mRNAs isolated from the encephalon, midbrain, and brainstem of 18-day-old rat embryos. In oocytes injected with encephalon, midbrain, or brainstem mRNAs, the Gly-current amplitudes (membrane current elicited by Gly; 1 mM Gly) were respectively 115 +/- 35, 346 +/- 28, and 389 +/- 22 nA, whereas the GABA-currents (1 mM GABA) were all < or =40 nA. Moreover, the Gly-currents desensitized faster in oocytes injected with encephalon or brainstem mRNAs. The EC(50) for Gly was 611 +/- 77 microM for encephalon, 661 +/- 28 microM for midbrain, and 506 +/- 18 microM for brainstem mRNA-injected oocytes, and the corresponding Hill coefficients were all approximately 2. Strychnine inhibited all of the Gly-currents, with an IC(50) of 56 +/- 3 nM for encephalon, 97 +/- 4 nM for midbrain, and 72 +/- 4 nM for brainstem mRNAs. During repetitive Gly applications, the Gly-currents were potentiated by 1.6-fold for encephalon, 2.1-fold for midbrain, and 1.3-fold for brainstem RNA-injected oocytes. Raising the extracellular Ca(2+) concentration significantly increased the Gly-currents in oocytes injected with midbrain and brainstem mRNAs. Reverse transcription-PCR studies showed differences in the Gly receptor (GlyR) alpha-subunits expressed, whereas the beta-subunit was present in all three types of mRNA. These results indicate differential expression of GlyR mRNAs in the brain areas examined, and these mRNAs lead to the expression of GlyRs that have different properties. The modulation of GlyRs by Ca(2+) could play important functions during brain development.
对从18日龄大鼠胚胎的脑、中脑和脑干分离的大鼠mRNA注射到非洲爪蟾卵母细胞中所表达的甘氨酸(Gly)和γ-氨基丁酸(GABA)受体进行了研究。在注射脑、中脑或脑干mRNA的卵母细胞中,甘氨酸电流幅度(由1 mM甘氨酸引起的膜电流)分别为115±35、346±28和389±22 nA,而GABA电流(1 mM GABA)均≤40 nA。此外,在注射脑或脑干mRNA的卵母细胞中,甘氨酸电流脱敏更快。注射脑mRNA的卵母细胞中甘氨酸的半数有效浓度(EC50)为611±77 μM,中脑为661±28 μM,脑干mRNA注射的卵母细胞为506±18 μM,相应的希尔系数均约为2。士的宁抑制所有甘氨酸电流,注射脑mRNA的IC50为56±3 nM,中脑为97±4 nM,脑干mRNA为72±4 nM。在重复应用甘氨酸期间,注射脑mRNA的卵母细胞中甘氨酸电流增强1.6倍,中脑增强2.1倍,脑干RNA注射的卵母细胞增强1.3倍。提高细胞外Ca2+浓度显著增加了注射中脑和脑干mRNA的卵母细胞中的甘氨酸电流。逆转录聚合酶链反应研究显示所表达的甘氨酸受体(GlyR)α亚基存在差异,但β亚基在所有三种类型的mRNA中均存在。这些结果表明在所研究的脑区中GlyR mRNA存在差异表达,并且这些mRNA导致具有不同特性的GlyR的表达。Ca2+对GlyR的调节可能在脑发育过程中发挥重要作用。