Paczkowski Filip A, Bryan-Lluka Lesley J
Department of Physiology and Pharmacology, School of Biomedical Sciences, The University of Queensland, Brisbane 4072, Queensland, Australia.
Naunyn Schmiedebergs Arch Pharmacol. 2002 Apr;365(4):312-7. doi: 10.1007/s00210-002-0528-z. Epub 2002 Feb 20.
It has been suggested from a previous study in our laboratory that differences in the pharmacology of the species variants of the noradrenaline transporter (NET) are the result of four non-conservative amino acid exchanges from the total of 26 amino acids that are divergent between the rat NET (rNET) and human NET (hNET). The aim of this study was to examine the effects of changing the rNET at each of these four amino acid residues, which markedly alter local charge distribution, to the amino acid found in hNET.Site-directed mutagenesis was used to create mutant cDNAs from rNET cDNA. The mutant NETs (rK7D, rE62K, rK375N and rR612Q), rNET and hNET were expressed in transiently transfected COS-7 cells to determine the effects of the mutations on the differing pharmacological properties of the species variants. The ratios of V(max) for noradrenaline uptake and B(max) for nisoxetine binding (which are a measure of the turnover number of the transporter, i.e. the number of transport cycles per min) were greater for rNET and rR612Q than for hNET, rK7D, rE62K and rK375N. The K(m) of noradrenaline was lower for hNET, rK7D, rE62K and rK375N than for rNET or rR612Q. There were no differences between the K(i) values for inhibition of noradrenaline uptake by nisoxetine for rNET, hNET or the mutants, but the K(i) values of cocaine were lower for hNET, rE62K and rR612Q than rNET or rK375N.Hence, the study showed that: (1) the aspartate 7, lysine 62 and asparagine 375 amino acid residues are important in determining the lower substrate translocation by hNET than rNET; (2) the aspartate 7 and lysine 62 residues in the N-terminus of hNET determine the higher affinities of substrates for the hNET than the rNET; and (3) the lysine 62 and glutamine 612 residues in the N- and C-termini, respectively, of hNET are determinants of the higher cocaine affinity for the hNET than rNET.
我们实验室之前的一项研究表明，去甲肾上腺素转运体（NET）物种变体在药理学上的差异，是大鼠NET（rNET）和人类NET（hNET）之间26个不同氨基酸中4个非保守氨基酸交换的结果。本研究的目的是研究将rNET中这四个显著改变局部电荷分布的氨基酸残基，替换为hNET中相应氨基酸后的影响。
利用定点诱变技术从rNET cDNA创建突变cDNA。将突变型NET（rK7D、rE62K、rK375N和rR612Q）、rNET和hNET在瞬时转染的COS-7细胞中表达，以确定这些突变对物种变体不同药理学特性的影响。rNET和rR612Q的去甲肾上腺素摄取V(max)与尼索西汀结合B(max)的比值（这是转运体周转数的一种衡量指标，即每分钟的转运循环数）高于hNET、rK7D、rE62K和rK375N。hNET、rK7D、rE62K和rK375N的去甲肾上腺素K(m)低于rNET或rR612Q。rNET、hNET或突变体对尼索西汀抑制去甲肾上腺素摄取的K(i)值没有差异，但hNET、rE62K和rR612Q的可卡因K(i)值低于rNET或rK375N。
因此，该研究表明：（1）天冬氨酸7、赖氨酸62和天冬酰胺375氨基酸残基对于决定hNET比rNET更低的底物转运很重要；（2）hNET N端的天冬氨酸7和赖氨酸62残基决定了底物对hNET的亲和力高于rNET；（3）hNET N端的赖氨酸62和C端的谷氨酰胺612残基分别是可卡因对hNET亲和力高于rNET的决定因素。
