Demchyshyn L L, Pristupa Z B, Sugamori K S, Barker E L, Blakely R D, Wolfgang W J, Forte M A, Niznik H B
Department of Psychiatry, University of Toronto, ON, Canada.
Proc Natl Acad Sci U S A. 1994 May 24;91(11):5158-62. doi: 10.1073/pnas.91.11.5158.
We report here on the isolation and characterization of a serotonin (5HT) transporter from Drosophila melanogaster. A 3.1-kb complementary DNA clone (dSERT) was found to encode a protein of 622 amino acid residues with a predicted molecular mass of approximately 69 kDa and a putative transmembrane topology characteristic of cloned members of the mammalian Na+/Cl- neurotransmitter cotransporter gene family. dSERT displays highest overall amino acid sequence identity with the mammalian 5HT (51%), norepinephrine (47%), and dopamine (47%) transporters and shares with all transporters 104 absolutely conserved amino acid residues. Upon transient expression in HeLa cells, dSERT exhibited saturable, high-affinity, and sodium-dependent [3H]5HT uptake with estimated Km and Vmax values of approximately 500 nM and 5.2 x 10(-18) mol per cell per min, respectively. In marked contrast to the human SERT (hSERT), 5HT-mediated transport by dSERT was not absolutely dependent on extracellular Cl-, while the sodium-dependent uptake of 5HT was facilitated by increased extracellular Cl- concentrations. dSERT displays a pharmacological profile and rank order of potency consistent with, but not identical to, mammalian 5HT transporters. Comparison of the affinities of various compounds for the inhibition of 5HT transport by both dSERT and hSERT revealed that antidepressants were 3- to 300-fold less potent on dSERT than on hSERT, while mazindol displayed approximately 30-fold greater potency for dSERT. Both cocaine and RTI-55 inhibited 5HT uptake by dSERT with estimated inhibition constants of approximately 500 nM, while high concentrations (> 10 microM) of dopamine, norepinephrine, octopamine, tyramine, and histamine failed to inhibit transport. In situ hybridization reveals the selective expression of dSERT mRNA to specific cell bodies in the ventral ganglion of the embryonic and larval Drosophila nervous system with a distribution pattern virtually identical to that of 5HT-containing neurons. The dSERT gene was mapped to position 60C on chromosome 2. The availability of the gene encoding the unique ion dependence and pharmacological characteristics of dSERT may allow for identification of those amino acid residues and structural motifs that confer the pharmacologic specificity and genetic regulation of the 5HT transport process.
我们在此报告从黑腹果蝇中分离并鉴定血清素（5HT）转运体的情况。一个3.1 kb的互补DNA克隆（dSERT）被发现编码一个由622个氨基酸残基组成的蛋白质，预测分子量约为69 kDa，具有哺乳动物Na⁺/Cl⁻神经递质共转运体基因家族克隆成员的推定跨膜拓扑结构特征。dSERT与哺乳动物的5HT转运体（51%）、去甲肾上腺素转运体（47%）和多巴胺转运体（47%）显示出最高的总体氨基酸序列同一性，并且与所有转运体共有104个绝对保守的氨基酸残基。在HeLa细胞中瞬时表达时，dSERT表现出可饱和、高亲和力且依赖钠的[³H]5HT摄取，估计的Km和Vmax值分别约为500 nM和5.2×10⁻¹⁸ mol/细胞/分钟。与人类SERT（hSERT）形成显著对比的是，dSERT介导的5HT转运并非绝对依赖细胞外Cl⁻，而细胞外Cl⁻浓度的增加促进了5HT的钠依赖性摄取。dSERT显示出与哺乳动物5HT转运体一致但不完全相同的药理学特征和效价顺序。比较各种化合物对dSERT和hSERT抑制5HT转运的亲和力发现，抗抑郁药对dSERT的效力比对hSERT低3至300倍，而吗茚酮对dSERT的效力约高30倍。可卡因和RTI - 55都抑制dSERT对5HT的摄取，估计抑制常数约为500 nM，而高浓度（>10 μM）的多巴胺、去甲肾上腺素、章鱼胺、酪胺和组胺未能抑制转运。原位杂交显示dSERT mRNA在胚胎和幼虫果蝇神经系统腹神经节的特定细胞体中选择性表达，其分布模式与含5HT的神经元几乎相同。dSERT基因被定位到2号染色体上的60C位置。编码具有独特离子依赖性和药理学特征的dSERT的基因的可得性，可能有助于鉴定赋予5HT转运过程药理学特异性和遗传调控的那些氨基酸残基和结构基序。
