Binding characteristics of levetiracetam to synaptic vesicle protein 2A (SV2A) in human brain and in CHO cells expressing the human recombinant protein
Binding characteristics of levetiracetam to synaptic vesicle protein 2A (SV2A) in human brain and in CHO cells expressing the human recombinant protein
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DOI：
10.1016/j.ejphar.2006.02.022
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发表时间：
2006-04-24
影响因子：
5
通讯作者：
Fuks, B
中科院分区：
文献类型：
--
作者：
Gillard, M;Chatelain, P;Fuks, B
抗癫痫药物左乙拉西坦（2S-（氧代-1-吡咯烷基）丁酰胺，Keppra（R））在大鼠脑中的特异性结合位点，被称为左乙拉西坦结合位点，几年前被发现。最近，该结合位点已被鉴定为突触囊泡蛋白2A（SV 2A），一种存在于突触囊泡中的蛋白[Lynch，B.，Lambeng，N，Nocka，K.，Kensel-Hammes，P.，Bajjalieh，S M.，Matagne，A.，去死吧，B，2004.突触囊泡蛋白SV 2A是抗癫痫药物左乙拉西坦的结合位点。Proc. Natl. Acad. Sci. USA，101，9861-9866.]。在这项研究中，我们的特点是结合特性的左乙拉西坦在死后人脑，并比较他们的人SV 2A在中国仓鼠卵巢（CHO）细胞中表达。结果显示，左乙拉西坦和[H-3]ucb 30889（一种以前被表征为大鼠脑中左乙拉西坦结合位点/SV 2A的合适配体的类似物）的结合特性[Gillard，M.，Fuks，B，Michel，P.，Vertongen，P.，马辛厄姆河Chatelain，P.，2003. [H-3]ucb 30889与大鼠脑内左乙拉西坦结合位点的结合特征。EUR. J. Pharmacol. 478，1-9.]，在人脑样品（大脑皮层、海马和小脑）和表达人SV 2A蛋白的CHO细胞膜中几乎相同。此外，该结果也类似于先前在大鼠脑中获得的结果。[H-3]ucb 30889在人脑中的结合和与SV 2A的结合是可饱和的和可逆的。在4 ℃时，其结合动力学最佳拟合，假设在所有组织中为两相模型。快速组件的半关联时间范围为1至2分钟，代表30%至36%的网站，而为慢组件的半关联时间范围为20至29分钟。在解离实验中，半关联时间为2至4分钟的快速组件（33%至49%的网站）和20至41分钟的缓慢组件。在表达SV 2A的人大脑皮层、海马、小脑和CHO细胞中，饱和结合曲线导致[H-3]ucb 30889的Kd值分别为53 +/- 7、55 +/- 9、70 +/- 11和75 +/- 33 nM。在所有脑区中计算出约3-4 pmol/mg蛋白质的B-max值。一些饱和曲线显示曲线Scatchard图，表明存在高和低亲和力结合位点。在这种情况下，计算出高亲和力位点（占总位点的24%至34%）的Kd值为25至30 nM，低亲和力位点的Kd值为200至275 nM。这在所有脑区和表达SV 2A蛋白的CHO细胞膜中观察到。不能通过[H-3]ucb 30889与SV 2B或C亚型的假定结合来解释，但可能反映了SV 2A糖基化的不同模式或SV 2A寡聚体的形成。进行竞争实验以确定多种化合物对SV 2A的亲和力，所述化合物包括左乙拉西坦、其一些类似物和已知与大鼠脑中的左乙拉西坦结合位点相互作用的其他分子，例如贝美格雷、戊四唑和利血平。我们发现在人脑、大鼠脑和表达人SV 2A的CHO细胞中测量的这些化合物的亲和力之间具有极好的相关性。总之，我们首次报告了人脑和大鼠脑中天然左乙拉西坦结合位点/SV 2A的结合特征与CHO细胞中表达的人重组SV 2A具有非常相似的特性（c）2006 Elsevier B. V.保留所有权利。
A specific binding site for the antiepileptic drug levetiracetam (2S-(oxo-1-pyrrolidinyl)butanamide, Keppra (R)) in rat brain, referred to as the levetiracetam binding site, was discovered several years ago. More recently, this binding site has been identified as the synaptic vesicle protein 2A (SV2A), a protein present in synaptic vesicles [Lynch, B., Lambeng, N, Nocka, K., Kensel-Hammes, P., Bajjalieh, S M., Matagne, A., Fuks, B., 2004. The synaptic vesicle protein SV2A is the binding site for the antiepileptic drug levetiracetam. Proc. Natl. Acad. Sci. USA, 101, 9861-9866.]. In this study, we characterized the binding properties of levetiracetam in post-mortem human brain and compared them to human SV2A expressed in Chinese hamster ovary (CHO) cells. The results showed that the binding properties of levetiracetam and [H-3]ucb 30889, an analogue that was previously characterized as a suitable ligand for levetiracetarn binding site/SV2A in rat brain [Gillard, M., Fuks, B, Michel, P., Vertongen, P., Massingham, R. Chatelain, P., 2003. Binding characteristics of [H-3]ucb 30889 to levetiracetam binding sites in rat brain. Eur. J. Pharmacol. 478, 1-9.], are almost identical in human brain samples (cerebral cortex, hippocampus and cerebellum) and in CHO cell membranes expressing the human SV2A protein. Moreover, the results are also similar to those previously obtained in rat brain. [H-3]ucb 30889 binding in human-brain and to SV2A was saturable and reversible. At 4 degrees C, its binding kinetics were best fitted assuming a two-phase model in all tissues. The half-times of association for the fast component ranged between 1 to 2 min and represent 30% to 36% of the sites whereas the half-times for the slow component ranged from 20 to 29 min. In dissociation experiments, the half-times were from 2 to 4 min for the fast component (33% to 49% of the sites) and 20 to 41 min for the slow component. Saturation binding curves led to K-d values for [H-3]ucb 30889 of 53 +/- 7, 55 +/- 9, 70 +/- 11 and 75 +/- 33 nM in human cerebral cortex, hippocampus, cerebellum and CHO cells expressing SV2A respectively. B-max values around 3-4 pmol/mg protein were calculated in all brain regions. Some of the saturation curves displayed curvilinear Scatchard plots indicating the presence of high and low affinity binding sites. When this was the case, K-d values from 25 to 30 nM for the high affinity sites (24% to 34% of total sites) and from 200 to 275 nM for the low affinity sites were calculated. This was observed in all brain regions and in CHO cell membranes expressing the SV2A protein. It cannot be explained by putative binding of [H-3]ucb 30889 to SV2B or C isoforms but may reflect different patterns of SV2A glycosylation or the formation of SV2A oligomers. Competition experiments were performed to determine the affinities for SV2A of a variety of compounds including levetiracetam, some of its analogues and other molecules known to interact with levetiracetam binding sites in rat brain such as bemegride, pentylenetetrazol and chlordiazepoxide. We found an excellent correlation between the affinities of these compounds measured in human brain, rat brain and CHO cells expressing human SV2A. In conclusion, we report for the first time that the binding characteristics of native levetiracetam binding sites/SV2A in human brain and rat brain share very similar properties with human recombinant SV2A expressed in CHO cells (c) 2006 Elsevier B.V. All rights reserved.