Plenge Per, Gether Ulrik, Rasmussen Søren G
Laboratory of Neuropsychiatry, Department of Neuroscience and Pharmacology, Rigshospitalet-6102, Blegdamsvej, DK-2100 Copenhagen, Denmark.
Eur J Pharmacol. 2007 Jul 12;567(1-2):1-9. doi: 10.1016/j.ejphar.2007.03.055. Epub 2007 Apr 14.
The human 5-HT transporter (hSERT) has two binding sites for 5-HT and 5-HT uptake inhibitors: the orthosteric high-affinity site and a low-affinity allosteric site. Activation of the allosteric site increases the dissociation half-life for some uptake inhibitors. The objectives of this study were 1) to identify hSERT mutations that inactivate the high-affinity site without affecting the allosteric site and 2) to observe allosteric effects in which hSERT binds R-citalopram with higher affinity than S-citalopram. Wild-type and mutant (Y95F, I172M, and Y95F/I172M) hSERTs were expressed in COS-7 cells, and their 5-HT uptake and uptake inhibitor-binding abilities were studied. The hSERT mutations did not alter affinities for 5-HT or paroxetine, but high-affinity binding of S-citalopram was severely affected, particularly by the I172M, and Y95F/I172M mutations - K(i) respectively 4 nM (wild-type), 35 nM, 1000 nM, and 17.100 nM (mutants). The allosteric site however, in wild-type hSERT and the three mutants was unaffected by the mutations as attenuation of the dissociation rate of the [(3)H]-paroxetine:hSERT complex in the presence of S-citalopram or paroxetine was the same for wild-type hSERT and the three mutants. Further, R-citalopram previously thought of as an inactive enantiomer strongly attenuated dissociation of the wild-type [(3)H]-imipramine:hSERT complex, whereas S-citalopram had almost no effect on this complex. These results suggest that 1: The allosteric site on hSERT is distinct from the site to which S-citalopram binds with high affinity. 2: The allosteric effects of R-citalopram on the dissociation of [(3)H]-imipramine from hSERT indicate that R-citalopram introduces a conformational change in hSERT.
人类5-羟色胺转运体（hSERT）有两个5-羟色胺和5-羟色胺摄取抑制剂的结合位点：正构高亲和力位点和低亲和力变构位点。变构位点的激活会增加某些摄取抑制剂的解离半衰期。本研究的目的是：1）鉴定使高亲和力位点失活而不影响变构位点的hSERT突变；2）观察hSERT以比S-西酞普兰更高的亲和力结合R-西酞普兰的变构效应。野生型和突变型（Y95F、I172M和Y95F/I172M）hSERT在COS-7细胞中表达，并研究了它们的5-羟色胺摄取和摄取抑制剂结合能力。hSERT突变未改变对5-羟色胺或帕罗西汀的亲和力，但S-西酞普兰的高亲和力结合受到严重影响，尤其是I172M和Y95F/I172M突变——抑制常数（K(i)）分别为4 nM（野生型）、35 nM、1000 nM和17100 nM（突变体）。然而，野生型hSERT和三个突变体中的变构位点不受突变影响，因为在存在S-西酞普兰或帕罗西汀的情况下，[(3)H]-帕罗西汀：hSERT复合物的解离速率衰减对于野生型hSERT和三个突变体是相同的。此外，之前被认为是无活性对映体的R-西酞普兰强烈减弱了野生型[(3)H]-丙咪嗪：hSERT复合物的解离，而S-西酞普兰对该复合物几乎没有影响。这些结果表明：1：hSERT上的变构位点与S-西酞普兰高亲和力结合的位点不同。2：R-西酞普兰对[(3)H]-丙咪嗪从hSERT解离的变构效应表明R-西酞普兰在hSERT中引入了构象变化。
