Mourot Alexandre, Kotzyba-Hibert Florence, Goeldner Maurice, Bamberg Ernst
Max Planck Institut für Biophysik, Frankfurt am Main, Germany.
J Mol Neurosci. 2006;30(1-2):3-4. doi: 10.1385/JMN:30:1:3.
Muscular and neuronal nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels and contain either two or five binding sites for acetylcholine (ACh). Binding of ACh molecules on the nAChR will trigger the fast opening of the channel and subsequent slow desensitization process. Neuronal alpha7 nicotinic receptors are made up of five identical subunits and possess five binding sites for ACh; this raises the question of how many sites must be occupied before channel opening. However, the effect of each ligand binding on gating is difficult to assess because of the reversible aspect of ligand binding at each site. One solution is to photochemically tether agonists to their binding sites. Such methodology has been applied elegantly and successfully on the homotetrameric cyclic-nucleotide-gated channels to evaluate the functional effects of each ligand binding on gating (Ruiz and Karpen, 1997). We therefore decided to develop a similar approach on Torpedo and neuronal alpha7 nAChRs with the photoactivatable agonist AC5 to investigate the effect of binding site occupancy on allosteric transitions of the receptor. In the dark, AC5 (see structure below) evokes robust currents on oocytes expressing Torpedo nAChR, displaying maximal amplitude comparable to ACh, with EC50 = 1.2 microM (Mourot et al., 2005). When the voltage-clamp oocyte was exposed to UV light in the presence of 30 microM AC5 for 50 s, there was a prolonged activation of the Torpedo nAChR, not reversible by washing, but inhibited by the noncompetitive blockers tetracaine and proadifen (see structure below). Both UV light and AC5 are required for this effect. However, further studies are required to determine whether the gradual decrease of the inward current reflects a slow desensitization process. AC5 is thus a potent photoactivatable agonist of the nAChR, which is able, upon UV irradiation, to incorporate covalently into the ACh-binding sites and to prolong activation of the nAChR. By extending this methodology to patch-clamp experiments, we will be able to incorporate one or several AC5s covalently into the muscular and neuronal nAChR at the single-channel level. Such study will help us understand the observed cooperative effect of gating and will contribute decisively to the controversial concerted vs sequential models for nAChR allosteric transitions.
肌肉型和神经型烟碱型乙酰胆碱受体（nAChRs）是五聚体配体门控离子通道，含有两个或五个乙酰胆碱（ACh）结合位点。ACh分子与nAChR结合会触发通道快速开放以及随后的缓慢脱敏过程。神经型α7烟碱型受体由五个相同的亚基组成，具有五个ACh结合位点；这就引出了一个问题，即通道开放前必须有多少个位点被占据。然而，由于每个位点上配体结合的可逆性，很难评估每个配体结合对门控的影响。一种解决办法是用光化学方法将激动剂束缚在其结合位点上。这种方法已被巧妙且成功地应用于同四聚体环核苷酸门控通道，以评估每个配体结合对门控的功能影响（Ruiz和Karpen，1997年）。因此，我们决定用可光激活的激动剂AC5在电鳐和神经型α7 nAChRs上开发类似的方法，以研究结合位点占据情况对受体变构转变的影响。在黑暗中，AC5（见下图结构）在表达电鳐nAChR的卵母细胞上引发强大电流，显示出与ACh相当的最大振幅，EC50 = 1.2微摩尔（Mourot等人，2005年）。当在30微摩尔AC5存在下将电压钳制的卵母细胞暴露于紫外光50秒时，电鳐nAChR会出现长时间激活，冲洗后不可逆转，但可被非竞争性阻滞剂丁卡因和普罗地芬（见下图结构）抑制。这种效应需要紫外光和AC5两者。然而，需要进一步研究来确定内向电流逐渐减少是否反映了缓慢的脱敏过程。因此，AC5是nAChR一种有效的可光激活激动剂，在紫外照射下，它能够共价结合到ACh结合位点并延长nAChR的激活。通过将这种方法扩展到膜片钳实验，我们将能够在单通道水平将一个或几个AC5共价结合到肌肉型和神经型nAChR中。这样的研究将有助于我们理解观察到的门控协同效应，并将对关于nAChR变构转变的有争议的协同模型与顺序模型做出决定性贡献。
