BACKGROUND Neurochemicals like serotonin and dopamine play crucial roles in human cognitive and emotional functions. Vesicular monoamine transporter 1 (VMAT1) transports monoamine neurotransmitters, and its variant (136Thr) is associated with various psychopathological symptoms and reduced monoamine uptake relative to 136Ile. We previously showed that two human-specific amino acid substitutions (Glu130Gly and Asn136Thr/Ile) of VMAT1 were subject to positive natural selection. However, the potential functional alterations caused by these substitutions (Glu130Gly and Asn136Thr) remain unclear. To assess functional changes in VMAT1 from an evolutionary perspective, we reconstructed ancestral residues and examined the role of these substitutions in monoamine uptake in vitro using fluorescent false neurotransmitters (FFN), which are newly developed substances used to quantitatively assay VMATs. RESULTS Immunoblotting confirmed that all the transfected YFP-VMAT1 variants are properly expressed in HEK293T cells at comparable levels, and no significant difference was seen in the density and the size of vesicles among them. Our fluorescent assays revealed a significant difference in FFN206 uptake among VMAT1 variants: 130Glu/136Asn, 130Glu/136Thr, and 130Gly/136Ile showed significantly higher levels of FFN206 uptake than 130Gly/136Asn and 130Gly/136Thr, indicating that both 130Glu and 136Ile led to increased neurotransmitter uptake, for which 136Thr and 136Asn were comparable by contrast. CONCLUSIONS These findings suggest that monoamine uptake by VMAT1 initially declined (from 130Glu/136Asn to 130Gly/136Thr) in human evolution, possibly resulting in higher susceptibility to the external environment of our ancestors.

中文翻译：

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VMAT1中人类特异性突变赋予单胺电路调节功能性变化和多方向进化。

背景技术诸如5-羟色胺和多巴胺的神经化学物质在人类的认知和情感功能中起着至关重要的作用。泡状单胺转运蛋白1（VMAT1）转运单胺神经递质，其变体（136Thr）与多种心理病理症状相关，相对于136Ile而言单胺摄取减少。我们先前显示，VMAT1的两个人类特异性氨基酸取代（Glu130Gly和Asn136Thr / Ile）经历了积极的自然选择。但是，由这些取代（Glu130Gly和Asn136Thr）引起的潜在功能改变仍不清楚。为了从进化的角度评估VMAT1的功能变化，我们重建了祖先残基，并使用荧光假神经递质（FFN）在体外检查了这些取代在单胺摄取中的作用，这是用于定量分析VMAT的新开发物质。结果免疫印迹证实，所有转染的YFP-VMAT1变体均在HEK293T细胞中以可比较的水平正确表达，并且它们之间的囊泡密度和大小均无显着差异。我们的荧光分析揭示了VMAT1变体之间FFN206摄取的显着差异：130Glu / 136Asn，130Glu / 136Thr和130Gly / 136Ile显示的FFN206摄取水平明显高于130Gly / 136Asn和130Gly / 136Thr，表明130Glu和136Ile均导致相比之下，神经递质的摄取增加了，而136Thr和136Asn却是可比的。结论这些发现表明，在人类进化过程中，VMAT1对单胺的吸收开始下降（从130Glu / 136Asn降至130Gly / 136Thr），