Chen N H, Ding J H, Wang Y L, Reith M E
Department of Pharmacology, Nanjing Medical University School of Basic Medical Sciences, People's Republic of China.
J Neurochem. 1997 May;68(5):1968-81. doi: 10.1046/j.1471-4159.1997.68051968.x.
The present study examines the interaction of Na+ and K+ with the binding of the cocaine analogue 3beta-(4-[125I]iodophenyl)tropane-2beta-carboxylic acid isopropyl ester to dopamine transporters (DATs) in rat striatal synaptosomal membranes at 37 degrees C. The binding increases with [Na+] from 10 to 100 mM and decreases with higher [Na+]. The presence of K+ reduces the maximal stimulatory effect of Na+ and causes a nonlinear EC50 shift for Na+. K+ strongly inhibits the binding at low [Na+]. Increasing [Na+] produces a linear IC50 shift for K+. Saturation analysis indicates a single binding site changing its affinity for the radioligand depending on [K+]/[Na+] ratio in the assay buffer. A reduced Bmax was observed in the presence of 10 mM Na+ and 30 mM K+. Both high [Na+] and high [K+] accelerate the dissociation of the binding, and K+-induced acceleration was abolished by increasing [Na+]. Least squares model fitting of equilibrium data and kinetic analysis of dissociation rates reveal competitive interactions between Na+ and K+ at two sites allosterically linked on the DAT: One site mediates the stimulatory effect of Na+, and the other site involves the radioligand binding and the inhibitory effect of cations on the binding. Various uptake blockers and substrates, dopamine in particular, display reduced potency in inhibiting the binding at a higher [K+]/[Na+] ratio.
本研究检测了在37℃下，大鼠纹状体突触体膜中，Na⁺和K⁺与可卡因类似物3β-(4-[¹²⁵I]碘苯基)托烷-2β-羧酸异丙酯与多巴胺转运体（DATs）结合的相互作用。结合作用随[Na⁺]从10 mM增加到100 mM而增强，而在更高的[Na⁺]浓度下则降低。K⁺的存在降低了Na⁺的最大刺激作用，并导致Na⁺的EC50发生非线性变化。在低[Na⁺]时，K⁺强烈抑制结合。增加[Na⁺]会使K⁺的IC50呈线性变化。饱和分析表明存在一个单一结合位点，其对放射性配体的亲和力根据测定缓冲液中的[K⁺]/[Na⁺]比值而变化。在存在10 mM Na⁺和30 mM K⁺的情况下，观察到Bmax降低。高[Na⁺]和高[K⁺]均加速结合的解离，并且通过增加[Na⁺]可消除K⁺诱导的加速作用。平衡数据的最小二乘模型拟合和解离速率的动力学分析揭示了Na⁺和K⁺在DAT上两个变构连接位点之间的竞争性相互作用：一个位点介导Na⁺的刺激作用，另一个位点涉及放射性配体结合以及阳离子对结合的抑制作用。各种摄取阻滞剂和底物，特别是多巴胺，在较高的[K⁺]/[Na⁺]比值下抑制结合的效力降低。
