Helms Nick, Kowalski Maria, Illes Peter, Riedel Thomas
Rudolf Boehm Institute for Pharmacology and Toxicology, University of Leipzig, Leipzig, Germany.
PLoS One. 2013 Nov 1;8(11):e79213. doi: 10.1371/journal.pone.0079213. eCollection 2013.
P2X3 receptors (P2XRs), as members of the purine receptor family, are deeply involved in chronic pain sensation and therefore, specific, competitive antagonists are of great interest for perspective pain management. Heretofore, Schild plot analysis has been commonly used for studying the interaction of competitive antagonists and the corresponding receptor. Unfortunately, the steady-state between antagonist and agonist, as a precondition for this kind of analysis, cannot be reached at fast desensitizing receptors like P2X3R making Schild plot analysis inappropriate. The aim of this study was to establish a new method to analyze the interaction of antagonists with their binding sites at the rapidly desensitizing human P2X3R. The patch-clamp technique was used to investigate the structurally divergent, preferential antagonists A317491, TNP-ATP and PPADS. The P2X1,3-selective α,β-methylene ATP (α,β-meATP) was used as an agonist to induce current responses at the wild-type (wt) P2X3R and several agonist binding site mutants. Afterwards a Markov model combining sequential transitions of the receptor from the closed to the open and desensitized mode in the presence or absence of associated antagonist molecules was developed according to the measured data. The P2X3R-induced currents could be fitted correctly with the help of this Markov model allowing identification of amino acids within the binding site which are important for antagonist binding. In conclusion, Markov models are suitable to simulate agonist antagonist interactions at fast desensitizing receptors such as the P2X3R. Among the antagonists investigated, TNP-ATP and A317491 acted in a competitive manner, while PPADS was identified as a (pseudo)irreversible blocker.
P2X3受体（P2XRs）作为嘌呤受体家族的成员，与慢性疼痛感觉密切相关，因此，特异性竞争性拮抗剂对于未来的疼痛管理具有重要意义。迄今为止，Schild图分析常用于研究竞争性拮抗剂与相应受体的相互作用。不幸的是，对于像P2X3R这样快速脱敏的受体，无法达到拮抗剂和激动剂之间的稳态，而这是这种分析的前提条件，因此Schild图分析并不适用。本研究的目的是建立一种新方法，用于分析拮抗剂与快速脱敏的人P2X3R上其结合位点的相互作用。采用膜片钳技术研究结构不同的优先拮抗剂A317491、TNP - ATP和PPADS。P2X1,3选择性α,β - 亚甲基ATP（α,β - meATP）用作激动剂，以在野生型（wt）P2X3R和几个激动剂结合位点突变体上诱导电流反应。之后，根据测量数据建立了一个马尔可夫模型，该模型结合了受体在存在或不存在相关拮抗剂分子时从关闭状态到开放状态和脱敏状态的顺序转变。借助该马尔可夫模型可以正确拟合P2X3R诱导的电流，从而确定结合位点内对拮抗剂结合重要的氨基酸。总之，马尔可夫模型适用于模拟快速脱敏受体（如P2X3R）上的激动剂 - 拮抗剂相互作用。在所研究的拮抗剂中，TNP - ATP和A317491以竞争性方式起作用，而PPADS被确定为（伪）不可逆阻滞剂。
