Function and expression of a splicing variant of vesicular glutamate transporter 1.
Function and expression of a splicing variant of vesicular glutamate transporter 1.
复制标题
囊泡谷氨酸转运蛋白1剪接变体的功能和表达。
DOI：
10.1016/j.bbamem.2017.02.002
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
Hiasa M
中科院分区：
文献类型：
--
作者：
Moriyama S;Iharada M;Omote H;Moriyama Y;Hiasa M
囊泡谷氨酸转运体（VGLUT）是一种负责谷氨酸在突触囊泡中囊泡储存的活性转运体，在谷氨酸能神经传递中起重要作用。VGLUT由VGLUT 1、VGLUT 2和VGLUT 3三种亚型组成。VGLUT1变体VGLUT1v具有来自外显子2和3之间的第二个内含子的额外75个碱基对序列，其对应于VGLUT1的第一环中的25个氨基酸残基，是VGLUT1中唯一的剪接变体，尽管VGLUT1v蛋白是否实际上在蛋白质水平上被翻译仍然未知。在本研究中，VGLUT1v在昆虫细胞中表达，溶解，纯化至接近同质，并检查其运输活性。含有纯化的VGLUT 1v的蛋白脂质体显示在施加内部正膜电位（Δ E）时积累谷氨酸。Δ Glu驱动的谷氨酸摄取活性需要Cl−，其药理学特征和动力学与其他VGLUT相当。视网膜膜含有两个VGLUT1部分，表观分子量为65和57 kDa。VGLUT1v特异性抗体对插入的25个氨基酸残基序列确定了一个65 kDa的免疫反应性多肽。免疫组织化学分析表明，VGLUT1v免疫反应性存在于感光细胞，并与突触囊泡。VGLUT1v免疫反应性也存在于松果体细胞中，但不存在于其他区域，包括大脑。这些结果表明，VGLUT1v在大鼠感光细胞中以功能状态存在，并参与神经递质的化学传递。
Vesicular glutamate transporter (VGLUT) is an active transporter responsible for vesicular storage of glutamate in synaptic vesicles and plays an essential role in glutamatergic neurotransmission. VGLUT consists of three isoforms, VGLUT1, VGLUT2, and VGLUT3. The VGLUT1 variant, VGLUT1v, with an additional 75-base pair sequence derived from a second intron between exons 2 and 3, which corresponds to 25 amino acid residues in the 1st loop of VGLUT1, is the only splicing variant among VGLUTs, although whether VGLUT1v protein is actually translated at the protein level remains unknown. In the present study, VGLUT1v was expressed in insect cells, solubilized, purified to near homogeneity, and its transport activity was examined. Proteoliposomes containing purified VGLUT1v were shown to accumulate glutamate upon imposition of an inside-positive membrane potential (Δψ). The Δψ-driven glutamate uptake activity requires Cl−and its pharmacological profile and kinetics are comparable to those of other VGLUTs. The retinal membrane contained two VGLUT1 moieties with apparent molecular masses of 65 and 57 kDa. VGLUT1v-specific antibodies against an inserted 25-amino acid residue sequence identified a 65-kDa immunoreactive polypeptide. Immunohistochemical analysis indicated that VGLUT1v immunoreactivity is present in photoreceptor cells and is associated with synaptic vesicles. VGLUT1v immunoreactivity is also present in pinealocytes, but not in other areas, including the brain. These results indicated that VGLUT1v exists in a functional state in rat photosensitive cells and is involved in glutamatergic chemical transmission.