Stein E, Cox J A, Seeburg P H, Verdoorn T A
Department of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232-6600.
Mol Pharmacol. 1992 Nov;42(5):864-71.
The pharmacological properties of two glutamate receptor subtypes, GluR-A/B and GluR-B/D, were examined in RNA-injected Xenopus oocytes using two-electrode voltage clamp. Concentration-response relations revealed that the potencies of L-glutamate, kainate, and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) varied slightly between the two receptor subtypes, but the rank order of agonist potency did not. The EC50 values for GluR-A/B receptors were 3.31 microM for AMPA, 6.16 microM for glutamate, and 57.5 microM for kainate, whereas the EC50 values for GluR-B/D receptors were 5.01 microM, 32.3 microM, and 64.6 microM for AMPA, L-glutamate, and kainate, respectively. The potencies of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX) were quantified by Schild analysis. The potency of NBQX at blocking currents mediated by GluR-A/B receptors changed depending on the agonist used to activate the receptors (pA2 values were as follows: for block of kainate, 7.23 +/- 0.01; L-glutamate, 6.78 +/- 0.02; AMPA, 6.95 +/- 0.02). Differences between agonists were less marked in cells expressing GluR-B/D receptors (pA2 values: kainate, 7.28 +/- 0.01; L-glutamate, 7.30 +/- 0.02; AMPA, 7.35 +/- 0.01). In each case, the slope of the Schild regression was not different from unity, consistent with competitive antagonism of these receptors by NBQX. CNQX also blocked GluR-A/B and GluR-B/D receptors competitively but was less potent than NBQX and did not differentiate between agonists or subunit combination. These data suggest that L-glutamate, kainate, and AMPA bind to different receptor substructures on recombinant AMPA receptors and that NBQX but not CNQX binds to these sites with different affinities. Moreover, because the properties of these binding sites vary between GluR-A/B and GluR-B/D receptors, our findings provide a basis for mutational analysis aimed at identifying receptor domains involved in agonist and antagonist binding.
利用双电极电压钳技术，在注射了RNA的非洲爪蟾卵母细胞中检测了两种谷氨酸受体亚型GluR - A/B和GluR - B/D的药理学特性。浓度 - 反应关系表明，L - 谷氨酸、海人酸和α - 氨基 - 3 - 羟基 - 5 - 甲基 - 4 - 异恶唑丙酸（AMPA）在这两种受体亚型之间的效价略有不同，但激动剂效价的排序顺序没有变化。GluR - A/B受体对AMPA、谷氨酸和海人酸的半数有效浓度（EC50）值分别为3.31微摩尔/升、6.16微摩尔/升和57.5微摩尔/升，而GluR - B/D受体对AMPA、L - 谷氨酸和海人酸的EC50值分别为5.01微摩尔/升、32.3微摩尔/升和64.6微摩尔/升。通过Schild分析对6 - 氰基 - 7 - 硝基喹喔啉 - 2,3 - 二酮（CNQX）和2,3 - 二羟基 - 6 - 硝基 - 7 - 氨磺酰基 - 苯并(f)喹喔啉（NBQX）的效价进行了定量。NBQX阻断由GluR - A/B受体介导的电流的效价取决于用于激活受体的激动剂（pA2值如下：阻断海人酸时为7.23±0.01；L - 谷氨酸时为6.78±0.02；AMPA时为6.95±0.02）。在表达GluR - B/D受体的细胞中，激动剂之间的差异不太明显（pA2值：海人酸为7.28±0.01；L - 谷氨酸为7.30±0.02；AMPA为7.35±0.01）。在每种情况下，Schild回归的斜率与1无差异，这与NBQX对这些受体的竞争性拮抗作用一致。CNQX也竞争性地阻断GluR - A/B和GluR - B/D受体，但效力低于NBQX，并且不能区分激动剂或亚基组合。这些数据表明，L - 谷氨酸、海人酸和AMPA与重组AMPA受体上不同的受体亚结构结合，并且NBQX而非CNQX以不同的亲和力结合这些位点。此外，由于这些结合位点的特性在GluR - A/B和GluR - B/D受体之间有所不同，我们的发现为旨在鉴定参与激动剂和拮抗剂结合的受体结构域的突变分析提供了基础。
