Carignano Camila, Barila Esteban Pablo, Spitzmaul Guillermo
Instituto de Investigaciones Bioquímicas de Bahía Blanca, Universidad Nacional del Sur-Consejo Nacional de Investigaciones Científicas y Técnicas, B8000FWB Bahía Blanca, Argentina.
Instituto de Investigaciones Bioquímicas de Bahía Blanca, Universidad Nacional del Sur-Consejo Nacional de Investigaciones Científicas y Técnicas, B8000FWB Bahía Blanca, Argentina.
Biochim Biophys Acta. 2016 Sep;1858(9):1964-1973. doi: 10.1016/j.bbamem.2016.05.019. Epub 2016 May 24.
The neuronal nicotinic acetylcholine receptor α4β2 forms pentameric proteins with two alternate stoichiometries. The high-sensitivity receptor is related to (α4)2(β2)3 stoichiometry while the low-sensitivity receptor to (α4)3(β2)2 stoichiometry. Both subtypes share two binding sites at the α4((+))/β2((-)) interface with high affinity for agonists. (α4)3(β2)2 has an additional binding site at the α4((+))/α4((-)) interface with low affinity for agonists. We investigated activation kinetics of both receptor subtypes by patch-clamp recordings of single-channel activity in the presence of several concentrations of acetylcholine (0.5 to 300μM). We used kinetic software to fit these data with kinetic models. We found that the high-sensitivity subtype correlates with the low-conductance channel (g-70=29pS) and does not activate with high efficacy. On the contrary, the low-sensitivity subtype correlated with a high-conductance channel (g-70=44pS) and exhibited higher activation efficacy. Opening events of individual nAChRs at high agonist concentrations occurred in clusters, which allowed us to determine kinetic constants for the activation of the triliganded receptor. Our kinetic modeling identified an intermediate state, between resting and open conformation of the receptor. Binding of the third molecule increases the efficacy of receptor activation by favoring the transition between resting and intermediate state around 18 times. The low rate for this transition in the diliganded receptor explains the action of acetylcholine as partial agonist when it binds to the high-affinity sites. The presence of the third binding site emerges as a potent modulator of nicotinic receptor α4β2 activation which may display different functions depending on agonist concentration.
神经元烟碱型乙酰胆碱受体α4β2形成具有两种交替化学计量的五聚体蛋白。高敏感性受体与(α4)2(β2)3化学计量相关，而低敏感性受体与(α4)3(β2)2化学计量相关。两种亚型在α4(+) /β2(-)界面共享两个对激动剂具有高亲和力的结合位点。(α4)3(β2)2在α4(+) /α4(-)界面有一个对激动剂低亲和力的额外结合位点。我们通过在几种乙酰胆碱浓度（0.5至300μM）存在下对单通道活性进行膜片钳记录来研究两种受体亚型的激活动力学。我们使用动力学软件将这些数据与动力学模型进行拟合。我们发现高敏感性亚型与低电导通道（g-70 = 29pS）相关，并且不能高效激活。相反，低敏感性亚型与高电导通道（g-70 = 44pS）相关，并表现出更高的激活效力。在高激动剂浓度下单个nAChRs的开放事件成簇发生，这使我们能够确定三配体受体激活的动力学常数。我们的动力学模型确定了受体静止和开放构象之间的中间状态。第三个分子的结合通过促进静止和中间状态之间约18倍的转变来增加受体激活的效力。双配体受体中这种转变的低速率解释了乙酰胆碱与高亲和力位点结合时作为部分激动剂的作用。第三个结合位点的存在成为烟碱型受体α4β2激活的有效调节剂，其可能根据激动剂浓度显示不同的功能。
