Department of Biological Chemistry, Alexander A. Silberman Institute of Life Sciences, Hebrew University of Jerusalem, 91904 Jerusalem, Israel.
J Biol Chem. 2010 Feb 12;285(7):5076-84. doi: 10.1074/jbc.M109.081216. Epub 2009 Dec 10.
The vesicular neurotransmitter transporter VMAT2 is responsible for the transport of monoamines into synaptic and storage vesicles. VMAT2 is the target of many psychoactive drugs and is essential for proper neurotransmission and survival. Here we describe a new expression system in Saccharomyces cerevisiae that takes advantage of the polyspecificity of VMAT2. Expression of rVMAT2 confers resistance to acriflavine and to the parkinsonian toxin 1-methyl-4-phenylpyridinium (MPP(+)) by their removal into the yeast vacuole. This expression system allowed identification of a new substrate, acriflavine, and isolation of mutants with modified affinity to tetrabenazine (TBZ), a non-competitive inhibitor of VMAT2 that is used in the treatment of various movement disorders including Tourette syndrome and Huntington chorea. Whereas one type of mutant obtained displayed decreased affinity to TBZ, a second type showed only a slight decrease in the affinity to TBZ, displayed a higher K(m) to the neurotransmitter serotonin, but conferred increased resistance to acriflavine and MPP(+). A protein where both types of mutations were combined (with only three amino acid replacements) lost most of the properties of the neurotransmitter transporter (TBZ-insensitive, no transport of neurotransmitter) but displayed enhanced resistance to the above toxicants. The work described here shows that in the case of rVMAT2, loss of traits acquired in evolution of function (such as serotonin transport and TBZ binding) bring about an improvement in older functions such as resistance to toxic compounds. A process that has taken millions of years of evolution can be reversed by three mutations.
囊泡神经递质转运体 VMAT2 负责将单胺类物质转运到突触小泡和储存小泡中。VMAT2 是许多精神活性药物的靶标,对正常的神经传递和生存至关重要。在这里,我们描述了一种新的酿酒酵母表达系统,该系统利用了 VMAT2 的多特异性。rVMAT2 的表达赋予了吖啶黄素和帕金森病毒素 1-甲基-4-苯基吡啶(MPP(+))的抗性,因为它们被转运到酵母液泡中。该表达系统鉴定了一种新的底物吖啶黄素,并分离到了与四苯嗪(TBZ)亲和力改变的突变体,TBZ 是 VMAT2 的非竞争性抑制剂,用于治疗各种运动障碍,包括妥瑞氏症和亨廷顿舞蹈症。一种获得的突变体显示出对 TBZ 的亲和力降低,而第二种突变体仅对 TBZ 的亲和力略有降低,对神经递质血清素的 K(m)值升高,但对吖啶黄素和 MPP(+)的抗性增加。同时具有这两种类型突变的蛋白质(仅发生三个氨基酸替换)丧失了大部分神经递质转运体的特性(对 TBZ 不敏感,不转运神经递质),但对上述有毒物质的抗性增强。这里描述的工作表明,在 rVMAT2 的情况下,功能进化中丧失的特性(如血清素转运和 TBZ 结合)会改善旧的功能,如对有毒化合物的抗性。一个经过数百万年进化的过程可以通过三个突变来逆转。