Rovira J C, Vicente-Agulló F, Campos-Caro A, Criado M, Sala F, Sala S, Ballesta J J
Instituto de Neurociencias, Universidad Miguel Hernández, San Juan de Alicante, Spain.
Pflugers Arch. 1999 Dec;439(1-2):86-92. doi: 10.1007/s004249900143.
Previous studies have shown that the gating mechanism of alpha3beta4 neuronal nicotinic receptors is affected by a residue in the middle of the M2-M3 loop of the beta4 subunit. We have extended the study of the same location to the alpha3 subunit. Bovine alpha3beta4 receptors were mutated in position 268, substituting the residue present in wild-type receptors, i.e. leucine in alpha3 and asparagine in beta4, for an aspartate. Wild-type and mutated alpha3 and beta4 subunits were combined to form four different receptors. We have measured macroscopic currents in Xenopus oocytes elicited by nicotine, and related them to surface receptor expression measured with an epibatidine-binding essay. We also obtained single-channel recordings of the receptors to study their kinetic behaviour. The results were analysed in terms of an allosteric model with three states. We found that the effect of the mutation in the alpha3 subunit on the gating of the receptor was similar to the corresponding mutation in the beta4 subunit. The effect when both subunits were mutated was additive, suggesting that the contribution of each subunit to the gating mechanism is independent.
先前的研究表明，α3β4神经元烟碱型受体的门控机制受β4亚基M2-M3环中间一个残基的影响。我们将对同一位置的研究扩展到了α3亚基。牛α3β4受体在第268位发生突变，将野生型受体中的残基，即α3中的亮氨酸和β4中的天冬酰胺替换为天冬氨酸。将野生型和突变型α3及β4亚基组合，形成四种不同的受体。我们测量了爪蟾卵母细胞中由烟碱引发的宏观电流，并将其与用埃博霉素结合试验测得的表面受体表达相关联。我们还获得了这些受体的单通道记录，以研究它们的动力学行为。结果根据具有三种状态的变构模型进行分析。我们发现α3亚基中的突变对受体门控的影响与β4亚基中的相应突变相似。当两个亚基都发生突变时，这种影响是累加的，这表明每个亚基对门控机制的贡献是独立的。
