Structural Studies of Vesicular Monoamine Transporters

描述(申请人提供)：囊泡单胺转运体2(VMAT2)是一种完整的膜蛋白，通过补充突触前囊泡中耗尽的神经递质储存，在胺能突触传递中发挥关键作用。它的工作原理是利用V型ATPase产生的预先存在的质子梯度，将一个细胞质阳离子胺的摄取与两个管腔质子的外流相结合。底物包括5-羟色胺、多巴胺、去甲肾上腺素、肾上腺素和组胺。VMAT2的重要性在于它将底物浓缩到突触小泡内的惊人水平(~500 mm)，以及它在将几种有毒化合物从神经元细胞质中隔离出来的积极作用。目前开出的治疗药物包括利血平[RES]和四苯肼[TBZ]。RES被用作治疗高血压和精神病的二线药物；TBZ被批准用于治疗亨廷顿病的运动失控和多发性抽动症的抽搐。尽管有这样的临床和药理学意义，但对转运、离子依赖、底物识别和抑制的结构基础知之甚少。VMAT2的晶体结构将提供关键信息，并可能给该领域带来革命性的变化。这项建议的目标是收集这一追求所需的初步数据：表达、纯化和获得真核细胞VMAT2同系物的衍射级晶体。

DESCRIPTION (provided by applicant): Vesicular monoamine transporter 2 (VMAT2) is an integral membrane protein that plays a critical role in aminergic synaptic transmission by replenishing depleted neurotransmitter stores in presynaptic vesicles. It works by harnessing a pre-existing proton gradient generated by V-type ATPase to couple the uptake of one cytoplasmic cationic amine with the efflux of two luminal protons. Substrates include serotonin, dopamine, norepinephrine, epinephrine, and histamine. The significance of VMAT2 is underscored by the phenomenal levels (~500 mM) to which it concentrates its substrates inside the synaptic vesicle and by its active role in sequestering several toxic compounds from the neuronal cytoplasm. Currently-prescribed therapeutics include the inhibitors reserpine [RES] and tetrabenazine [TBZ]. RES is used as a second-line agent to treat hypertension and psychosis; TBZ is approved to treat the uncontrolled movements in Huntington's disease and the tics in Tourette's syndrome. Despite such clinical and pharmacological significance, very little is known about the structural basis for transport, ion-dependence, substrate recognition, and inhibition. A VMAT2 crystal structure would provide the crucial information and likely revolutionize the field. The goal of this proposal is to gather the preliminary data required for such a pursuit: to express, purify, and obtain diffraction-quality crystals of a eukaryotic VMAT2 homologue.