TARGETING ALPHA7 NACHR FOR THERAPEUTICS EFFECTS

α - 7神经元烟碱乙酰胆碱受体（AchR）亚型可能是一个重要的治疗靶点，在治疗阿尔茨海默病、中风和精神分裂症方面具有潜在的意义。α 7亚型的激活已被证明具有细胞保护作用并增强突触传递，这可能是报道的尼古丁药物获得积极认知作用的基础。我们的数据表明，alpha7受体有两种不同的激活模式。通常，研究集中在相对较大的瞬态电流上，这种电流可以通过快速应用高浓度的激动剂来刺激。然而，在持续存在如此高的激动剂浓度时，几乎所有的稳态电流都被脱敏过程抑制，而在较低的激动剂浓度下，少量的稳态激活持续存在。我们关于受体的生物物理学和α 7选择性激动剂的细胞保护作用的初步数据表明，低浓度激动剂对这种钙渗透受体亚型的稳态激活可能是最具治疗意义的功能模式。因此，我们将详细研究alpha7受体的激活和脱敏特性，扩展我们对浓度/反应函数的分析，包括对稳态电流的分析。我们将使用内源性激活剂Ach和胆碱以及alpha7选择性部分激动剂HMBA进行单通道和全细胞膜片钳分析。我们将测试alpha7受体的生物物理特性模型，以提高alpha7受体的治疗靶向性。HMBA是DMXB （GTS-21）的活性代谢物，DMXB是一种刚刚进入阿尔茨海默病2期临床试验的药物。DMXB本身是α - 7选择性麻瓜碱衍生物家族的原型，我们已经证明，其中许多衍生物，如尼古丁，有两个阶段的作用，最初刺激受体，然后引起持久的抑制。我们将研究这种抑制活性的性质和分子相互作用的基础。我们将描述Ach和胆碱诱导的alpha7受体的脱敏状态，并确定应用DMXB和类似药物后观察到的残留抑制是否代表加速脱敏或替代形式的抑制。为了提高我们靶向alpha7受体的治疗能力，人类和大鼠alpha7受体的嵌合体将被制造出来。然后，通过使用对人类和大鼠α 7野生型受体的激活或抑制表现出选择性的试剂，将有可能确定调节激活和脱敏的蛋白质的结构元件。

The alpha7 neuronal nicotinic acetylcholine receptor (AchR) subtype may be an important therapeutic target, with potential significance for the treatment of Alzheimer's disease, stroke, and schizophrenia. Activation of the alpha7 subtype has been shown to have cytoprotective effects and enhance synaptic transmission, which may underlie reported positive cognitive effects obtained with nicotinic agents. Our data indicate that alpha7 receptors have two contrasting modes of activation. Typically, studies have focused on the relatively large transient currents that can be stimulated by rapid application of high concentrations of agonist. However, in the continued presence of such high agonist concentrations, virtually all steady-state current is suppressed by the desensitization process, while at lower agonist concentrations a small amount of steady-state activation persists. Our preliminary data regarding the biophysics of the receptor and the cytoprotective effects of alpha7-selective agonists indicate that steady-state activation of this calcium permeable receptor subtype by low agonist concentrations may represent the functional modality of greatest therapeutic significance. We will therefore study the activation and desensitization properties of alpha7 receptors in detail, expanding our analysis of concentration/response function to include an analysis of the steady-state currents. We will conduct single-channel and whole-cell patch-clamp analysis, using the endogenous activators, Ach and choline, as well as the alpha7-selective partial agonist HMBA. We will test models of the biophysical properties of alpha7 receptors, towards the goal of improving therapeutic targeting of alpha7 receptors. HMBA is the active metabolite of DMXB (GTS-21), a drug just entering phase 2 clinical trials for Alzheimer's disease. DMXB itself is the prototype for a family of alpha7-selective anabaseine derivatives, many of which we have shown, like nicotine, have two phases of action, initially stimulating the receptor, then subsequently causing a long lasting inhibition. We will investigate the nature of this inhibitory activity and the molecular interactions which underlie it. We will characterize the desensitized states of the alpha7 receptor induced by Ach and choline, and determine whether the residual inhibition observed after the application of DMXB and similar agents represents accelerated desensitization or alternative forms of inhibition. In order to improve our ability to target alpha7 receptors for therapeutics, chimeras of the human and rat alpha7 receptors will be made. Then, through the use of agents which show selectivity for the activation or inhibition of the human and rat alpha7 wild-type receptors, it will be possible to identify structural elements of the proteins that regulate activation and desensitization.