Eshleman A J, Calligaro D O, Eldefrawi M E
Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore 21201.
Membr Biochem. 1993 Jul-Sep;10(3):129-44. doi: 10.3109/09687689309150260.
Sodium regulation of ligand binding to the dopamine transporter of rat and/or bovine striata was investigated using a filtration binding assay. In low Na+ phosphate or bicarbonate-buffered sucrose (300 mOsm), the tissue exhibited high affinity for [3H]cocaine which was reduced by the addition of Na+ in a dose-dependent manner. However, [3H]GBR 12935 binding was insensitive to Na+ in these physiological buffers. Although binding of [3H]GBR 12935 was displaced by cocaine in a manner consistent with competitive displacement, a non-linear affinity shift of the displacement of [3H]GBR 12935 by cocaine suggests that the two ligands bind to distinct sites. Binding of both radioligands was suppressed when measured in sodium-free 50 nM Tris-sucrose and increased with the addition of Na+. Scatchard analysis indicated that Bmax for [3H]cocaine binding in Tris plus 120 mM NaCl reached the same level as in the physiological buffers. In Krebs-Ringer buffer with phosphate, bicarbonate or Tris, which contained 120 nM NaCl, both [3H]cocaine and [3H]WIN 35428 binding exhibited lower affinities than in Na(+)-deficient phosphate buffer. It is suggested that the cation form of Tris binds to the dopamine transporter and that the Tris-receptor complex does not bind [3H]cocaine or [3H]GBR 12935. Na+ displaces Tris, forming a Na(+)-receptor complex which binds these ligands. Thus, it is suggested that the Na(+)-dependent binding of cocaine to the dopamine transporter is observed only in Tris.
采用过滤结合分析法研究了钠对大鼠和/或牛纹状体多巴胺转运体配体结合的调节作用。在低钠磷酸盐或碳酸氢盐缓冲的蔗糖溶液（300 mOsm）中，组织对[³H]可卡因表现出高亲和力，添加钠离子后，这种亲和力呈剂量依赖性降低。然而，在这些生理缓冲液中，[³H]GBR 12935的结合对钠离子不敏感。尽管[³H]GBR 12935的结合可被可卡因以竞争性置换的方式取代，但可卡因对[³H]GBR 12935置换的非线性亲和力变化表明这两种配体结合于不同位点。在无钠的50 nM Tris - 蔗糖溶液中测定时，两种放射性配体的结合均受到抑制，添加钠离子后结合增加。Scatchard分析表明，在Tris加120 mM NaCl中，[³H]可卡因结合的Bmax与在生理缓冲液中达到相同水平。在含有120 nM NaCl的磷酸盐、碳酸氢盐或Tris的Krebs - Ringer缓冲液中，[³H]可卡因和[³H]WIN 35428的结合亲和力均低于在缺钠磷酸盐缓冲液中的亲和力。提示Tris的阳离子形式与多巴胺转运体结合，且Tris - 受体复合物不结合[³H]可卡因或[³H]GBR 12935。钠离子取代Tris，形成结合这些配体的Na⁺ - 受体复合物。因此，提示仅在Tris中观察到可卡因与多巴胺转运体的Na⁺依赖性结合。
