Niu L, Vazquez R W, Nagel G, Friedrich T, Bamberg E, Oswald R E, Hess G P
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY 14853-2703, USA.
Proc Natl Acad Sci U S A. 1996 Nov 12;93(23):12964-8. doi: 10.1073/pnas.93.23.12964.
Xenopus laevis oocytes have been used extensively during the past decade to express and study neurotransmitter receptors of various origins and subunit composition and also to express and study receptors altered by site-specific mutations. Interpretations of the effects of structural differences on receptor mechanisms were, however, hampered by a lack of rapid chemical reaction techniques suitable for use with oocytes. Here we describe flow and photolysis techniques, with 2-ms and 100-microseconds time resolution, respectively, for studying neurotransmitter receptors in giant (approximately 20-microns diameter) patches of oocyte membranes, using muscle and neuronal acetylcholine receptors as examples. With these techniques, we find that the muscle receptor in BC3H1 cells and the same receptor expressed in oocytes have comparable kinetic properties. This finding is in contrast to previous studies and raises questions regarding the interpretations of the many studies of receptors expressed in oocytes in which an insufficient time resolution was available. The results obtained indicate that the rapid reaction techniques described here, in conjunction with the oocyte expression system, will be useful in answering many outstanding questions regarding the structure and function of diverse neurotransmitter receptors.
在过去十年中，非洲爪蟾卵母细胞被广泛用于表达和研究各种来源及亚基组成的神经递质受体，还用于表达和研究因位点特异性突变而改变的受体。然而，由于缺乏适用于卵母细胞的快速化学反应技术，对受体机制中结构差异影响的解读受到了阻碍。在此，我们描述了分别具有2毫秒和100微秒时间分辨率的流动和光解技术，以肌肉型和神经元型乙酰胆碱受体为例，用于研究卵母细胞膜大（直径约20微米）片膜中的神经递质受体。使用这些技术，我们发现BC3H1细胞中的肌肉型受体与在卵母细胞中表达的相同受体具有可比的动力学特性。这一发现与先前的研究形成对比，并对许多卵母细胞中表达的受体研究的解读提出了疑问，这些研究中时间分辨率不足。所获得的结果表明，本文所述的快速反应技术与卵母细胞表达系统相结合，将有助于回答许多关于各种神经递质受体结构和功能的悬而未决的问题。
