Substrate induced currents through the monoamine transporters and single cell uptake of the fluorescent substrate APP+

The concentrative power of the transporters for dopamine (DAT), norepinephrine (NET) and serotonin (SERT) is thought to be fueled by the transmembrane Na+ gradient, but it is conceivable that they can also tap other energy sources, e.g. membrane voltage and/or the transmembrane K+ gradient. We address this by recording uptake of endogenous substrates or the fluorescent substrate APP+ ((4-(4-dimethylamino)phenyl-1-methylpyridinium) under voltage control in cells expressing DAT, NET or SERT. We show that DAT and NET differ from SERT in intracellular handling of K+. In DAT and NET, substrate uptake was voltage-dependent due to the transient nature of intracellular K+ binding, which precluded K+ antiport. SERT, however, antiports K+ and achieves voltage-independent transport. Thus, there is a trade-off between maintaining constant uptake and harvesting membrane potential for concentrative power, which we conclude to occur due to subtle differences in the kinetics of co-substrate ion binding...人们普遍认为，多巴胺转运体（Dopamine Transporter, DAT）、去甲肾上腺素转运体（Norepinephrine Transporter, NET）与5-羟色胺转运体（Serotonin Transporter, SERT）的浓缩转运能力由跨膜钠离子梯度供能，但也有假说指出这类转运体可利用其他能量来源，例如膜电位或跨膜钾离子梯度。本研究通过在表达DAT、NET或SERT的细胞中采用电压钳控技术，记录内源性底物或荧光底物APP+（(4-(4-二甲氨基)苯基-1-甲基吡啶鎓)）的摄取过程，对该假说进行了验证。研究表明，DAT与NET在细胞内钾离子的处理机制上与SERT存在差异：对于DAT和NET而言，由于细胞内钾离子结合具有瞬时性，无法实现钾离子反向转运，因此底物摄取过程具有电压依赖性；而SERT则可介导钾离子反向转运，实现不依赖膜电位的底物转运。综上，维持底物摄取恒定与利用膜电位获取浓缩转运能量之间存在权衡关系，我们认为该现象源于共底物离子结合动力学的细微差异。