The dopamine transporter (DAT) is a transmembrane protein that terminates dopamine signaling in the brain by driving rapid dopamine reuptake into presynaptic nerve terminals. Several lines of evidence indicate that DAT dysfunction is linked to neuropsychiatric disorders such as attention-deficit/hyperactivity disorder (ADHD), bipolar disorder (BPD), and autism spectrum disorder (ASD). Indeed, individuals with these disorders have been found to express the rare, functional DAT coding variant Val559, which confers anomalous dopamine efflux (ADE) in vitro and in vivo. To elucidate the impact of the DAT Val559 variant on membrane diffusion dynamics, we implemented our antagonist-conjugated quantum dot (QD) labeling approach to monitor the lateral mobility of single particle-labeled transporters in transfected HEK-293 and SK-N-MC cells. Our results demonstrate significantly higher diffusion coefficients of DAT Val559 compared to those of DAT Ala559, effects likely determined by elevated N-terminal transporter phosphorylation. We also provide pharmacological evidence that PKCβ-mediated signaling supports enhanced DAT Val559 membrane diffusion rates. Additionally, our results are complimented with diffusion rates of phosphomimicked and phosphorylation-occluded DAT variants. Furthermore, we show DAT Val559 has a lower propensity for membrane clustering, which may be caused by a mutation-derived shift out of membrane microdomains leading to faster lateral membrane diffusion rates. These findings further demonstrate a functional impact of DAT Val559 and suggest that changes in transporter localization and lateral mobility may sustain ADE and contribute to alterations in dopamine signaling underlying multiple neuropsychiatric disorders.

中文翻译：

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单量子点成像揭示了注意力缺陷多动障碍/自闭症/双相情感障碍相关多巴胺转运蛋白变异体的膜扩散和聚集中 PKCβ 依赖性改变。

多巴胺转运蛋白 (DAT) 是一种跨膜蛋白，通过驱动突触前神经末梢快速重摄取多巴胺来终止大脑中的多巴胺信号传导。多项证据表明，DAT 功能障碍与注意力缺陷/多动障碍 (ADHD)、双向情感障碍 (BPD) 和自闭症谱系障碍 (ASD) 等神经精神疾病有关。事实上，已发现患有这些疾病的个体表达罕见的功能性 DAT 编码变体 Val559，该变体在体外和体内导致多巴胺流出 (ADE) 异常。为了阐明 DAT Val559 变体对膜扩散动力学的影响，我们实施了拮抗剂共轭量子点 (QD) 标记方法来监测转染的 HEK-293 和 SK-N-MC 细胞中单颗粒标记转运蛋白的横向迁移率。我们的结果表明，与 DAT Ala559 相比，DAT Val559 的扩散系数显着更高，这种效应可能是由 N 末端转运蛋白磷酸化升高决定的。我们还提供药理学证据表明 PKCβ 介导的信号传导支持增强 DAT Val559 膜扩散速率。此外，我们的结果还得到了磷酸化和磷酸化封闭 DAT 变体的扩散率的补充。此外，我们表明 DAT Val559 具有较低的膜聚集倾向，这可能是由于突变引起的膜微域转移导致更快的侧向膜扩散速率。这些发现进一步证明了 DAT Val559 的功能影响，并表明转运蛋白定位和横向移动性的变化可能维持 ADE 并导致多种神经精神疾病背后的多巴胺信号传导的改变。