Mechanistic analyses of ion dependences in a high-affinity human serotonin transport system in transfected murine fibroblast cells.
Mechanistic analyses of ion dependences in a high-affinity human serotonin transport system in transfected murine fibroblast cells.
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转染的鼠成纤维细胞中高亲和力人血清素转运系统中离子依赖性的机制分析。
DOI：
10.1111/j.1469-7793.1998.903bj.x
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Lam,DM
中科院分区：
文献类型：
--
作者：
Chang,AS;Lam,DM
1A克隆细胞系，L-S1，已经从人基因组DNA转染到培养的小鼠L-M成纤维细胞中鉴定出来。由于该转染细胞系稳定表达高亲和力5-羟色胺（5-HT）转运机制，其动力学和药理学性质与其他5-羟色胺摄取系统相当，因此将其用于研究Na+和Cl−离子参与该系统的配体结合和动力学摄取过程的机制。2当在低温（4 °C）下孵育时，能够定量评估丙咪嗪可置换的5-[3 H]HT与5-HT转运系统的结合。这种结合活性对5-HT受体亚型的各种特异性配体的存在不敏感。3 Imipramine-可置换的5-[3 H]HT与完整L-S1细胞的结合显示为Cl−-依赖性但Na+-非依赖性过程。氯离子缺乏促进配体结合的结合协同性。外部Cl−浓度的变化改变了结合的Kd，但没有改变结合的Bmax。4观察到总体转运活性高度依赖于外部Na+和Cl−浓度，其特征在于每个转运周期的5-HT：Na+：Cl−偶联比为1：1：1。Na+和Cl-离子外部浓度的变化仅改变了转运的K，而没有改变转运的Vmax。5结合和动力学结果均与动力学模型预测一致，即Cl-离子促进5-HT与转运系统结合，以及Na+离子使结合的5-HT能够跨质膜转运。因此，Na+和Cl−离子在运输循环中促进了机械上不同和可识别的功能。
1A clonal cell line, L‐S1, has been identified from transfection of human genomic DNA into cultured mouse L‐M fibroblasts. Because this transfectant cell line stably expresses a high‐affinity serotonin (5‐HT) transport mechanism with kinetic and pharmacological properties comparable to those of other serotonin uptake systems, it was used to investigate the mechanistic involvement of Na+and Cl−ions in the ligand binding and kinetic uptake processes of this system.2Intact transfectant cells, when incubated at low temperature (4 °C), enabled quantitative assessment of imipramine‐displaceable 5‐[3H]HT binding to the 5‐HT transport system. This binding activity is insensitive to the presence of various ligands specific for 5‐HT receptor subtypes.3Imipramine‐displaceable 5‐[3H]HT binding to intact L‐S1 cells was shown to be a Cl−‐dependent but Na+‐independent process. Chloride ions lack binding co‐operativity in facilitating ligand binding. Changes in external Cl−concentration altered theKdbut not theBmaxof binding.4The overall transport activity was observed to be highly dependent on both external Na+and Cl−concentrations, characterized by a 5‐HT:Na+:Cl−coupling ratio of 1:1:1 per transport cycle. Alterations in the external concentrations of both Na+and Cl−ions altered only theKmand not theVmaxof transport.5Both binding and kinetic results are consistent with kinetic modelling predictions of the Cl−ion in facilitating 5‐HT binding to the transport system, and of the Na+ion in enabling translocation of bound 5‐HT across the plasma membrane. Thus, Na+and Cl−ions facilitate mechanistically distinct and discernible functions in the transport cycle.