期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2002
卷号：99
期号：6
页码：3956-3961
DOI：10.1073/pnas.052699599
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Neuronal nicotinic acetylcholine (AcCho) receptors composed of 7-subunits (7-AcChoRs) are involved in many physiological activities. Nevertheless, very little is known about their single-channel characteristics. By using outside-out patch-clamp recordings from Xenopus oocytes expressing wild-type (wt) 7-AcChoRs, we identified two classes of channel conductance: a low conductance ({gamma}L) of 72 pS and a high one ({gamma}H) of 87 pS, with mean open-times ({tau}op) of 0.6 ms. The same classes of conductances, but longer {tau}op (3 ms), were seen in experiments with chimeric 7 receptors in which the wt7 extracellular C terminus was fused to the green fluorescent protein (wt7-GFP AcChoRs). In contrast, channels with three different conductances were gated by AcCho in oocytes expressing 7 receptors carrying a Leu-to-Thr 248 mutation (mut7) or oocytes expressing chimeric mut7-GFP receptors. These conductance levels were significantly smaller, and their mean open-times were larger, than those of wt7-AcChoRs. Interestingly, in the absence of AcCho, these oocytes showed single-channel openings of the same conductances, but shorter {tau}op, than those activated by AcCho. Accordingly, human homomeric wt7 receptors open channels of high conductance and brief lifetime, and fusion to GFP lengthens their lifetime. In contrast, mut7 receptors open channels of lower conductance and longer lifetime than those gated by wt7-AcChoRs, and these parameters are not greatly altered by fusing the mut7 to GFP. All this evidence shows that GFP-tagging can alter importantly receptor kinetics, a fact that has to be taken into account whenever tagged proteins are used to study their function.
