Li L B, Reith M E
Department of Biology, Illinois State University, Normal, USA.
J Neurochem. 1999 Mar;72(3):1095-109. doi: 10.1046/j.1471-4159.1999.0721095.x.
Although much is known about the effects of Na+, K+, and Cl- on the functional activity of the neuronal dopamine transporter, little information is available on their role in the initial event in dopamine uptake, i.e., the recognition step. This was addressed here by studying the inhibition by dopamine of the binding of [3H]WIN 35,428 [2beta-carbomethoxy-3beta-(4-fluorophenyl)[3H]tropane], a phenyltropane analogue of cocaine, to the cloned human dopamine transporter expressed in HEK-293 cells. The decrease in the affinity of dopamine (or WIN 35,428) binding affinity with increasing [K+] could be fitted to a competitive model involving an inhibitory cation site (1) overlapping with the dopamine (or WIN 35,428) domain. The K+ IC50 for inhibiting dopamine or WIN 35,428 binding increased linearly with [Na+], indicating a K(D,Na+) of 30-44 mM and a K(D,K+) of 13-16 mM for this cation site. A second Na+ site (2), distal from the WIN 35,428 domain but linked by positive allosterism, was indicated by model fitting of the WIN 35,428 binding affinities as a function of [Na+]. No strong evidence for this second site was obtained for dopamine binding in the absence or presence of low (20 mM) Cl- and could not be acquired for high [Cl-] because of the lack of a suitable substitute ion for Na+. The K(D) but not Bmax of [3H]WIN 35,428 binding increased as a function of the [K+]/[Na+] ratio regardless of total [Cl-] or ion tonicity. A similar plot was obtained for the Ki of dopamine binding, with Cl- at > or = 140 mM decreasing the Ki. At 290 mM Cl- and 300 mM Na+ the potency of K+ in inhibiting dopamine binding was enhanced as compared with the absence of Cl- in contrast to the lack of effect of Cl- up to 140 mM (Na up to 150 mM). The results indicate that Cl- at its extracellular level enhances dopamine binding through a mechanism not involving site 1. The observed correspondence between the WIN 35,428 and dopamine domains in their inclusion of the inhibitory cation site explains why many of the previously reported interrelated effects of Na+ and K+ on the binding site of radiolabeled blockers to the dopamine transporter are applicable to dopamine uptake in which dopamine recognition is the first step.
尽管人们对钠离子、钾离子和氯离子对神经元多巴胺转运体功能活性的影响已了解很多，但关于它们在多巴胺摄取初始事件即识别步骤中的作用却知之甚少。本文通过研究多巴胺对[3H]WIN 35,428（2β-甲氧羰基-3β-(4-氟苯基)[3H]托烷）与在HEK-293细胞中表达的克隆人多巴胺转运体结合的抑制作用来解决这一问题。随着[K+]增加，多巴胺（或WIN 35,428）结合亲和力的降低可拟合为一个竞争性模型，该模型涉及一个与多巴胺（或WIN 35,428）结构域重叠的抑制性阳离子位点(1)。抑制多巴胺或WIN 35,428结合的K+ IC50随[Na+]线性增加，表明该阳离子位点的K(D,Na+)为30 - 44 mM，K(D,K+)为13 - 16 mM。通过将WIN 35,428结合亲和力作为[Na+]的函数进行模型拟合，表明存在第二个钠离子位点(2)，它远离WIN 35,428结构域，但通过正构象效应相连。在不存在或存在低浓度(20 mM)氯离子的情况下，对于多巴胺结合没有获得关于这个第二位点的有力证据，并且由于缺乏合适的钠离子替代离子，在高[Cl-]时也无法获得相关证据。无论总[Cl-]或离子张力如何，[3H]WIN 35,428结合的K(D)而非Bmax随[K+]/[Na+]比值增加。多巴胺结合的Ki也得到类似的曲线，当Cl-≥140 mM时，Ki降低。在290 mM Cl-和300 mM Na+时，与不存在Cl-相比，K+抑制多巴胺结合的效力增强，而在Cl-浓度高达140 mM（Na浓度高达150 mM）时Cl-没有作用。结果表明，细胞外水平的氯离子通过一种不涉及位点1的机制增强多巴胺结合。在WIN 35,428和多巴胺结构域中观察到的包含抑制性阳离子位点的对应关系解释了为什么许多先前报道的钠离子和钾离子对放射性标记阻滞剂与多巴胺转运体结合位点的相互关联效应适用于多巴胺摄取，其中多巴胺识别是第一步。
