VESICULAR L-GLUTAMATE TRANSPORTER IN MICROVESICLES FROM BOVINE PINEAL GLANDS - DRIVING-FORCE, MECHANISM OF CHLORIDE ANION ACTIVATION, AND SUBSTRATE-SPECIFICITY
VESICULAR L-GLUTAMATE TRANSPORTER IN MICROVESICLES FROM BOVINE PINEAL GLANDS - DRIVING-FORCE, MECHANISM OF CHLORIDE ANION ACTIVATION, AND SUBSTRATE-SPECIFICITY
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DOI：
10.1074/jbc.270.38.22314
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发表时间：
1995-09-22
影响因子：
4.8
通讯作者：
YAMAMOTO, A
中科院分区：
文献类型：
--
作者：
MORIYAMA, Y;YAMAMOTO, A
松果体细胞是合成和分泌褪黑素的内分泌细胞，具有大量的含有L谷氨酸转运蛋白的突触样微泡(Moriyama，Y.和Yamamoto，A.(1995)FEBS Left，367,233-236)。本研究从牛松果体分离的MVS中对L谷氨酸转运体的驱动力、对阴离子的需求和底物专一性进行了研究。加入三磷酸腺苷后，MVS积累了L-谷氨酸。摄取显著依赖于囊外的氯离子浓度，无氯离子时可忽略不计，在25 mm处达到最大值，在20~100 mm处逐渐下降。膜电位在0-10 mM的氯离子浓度范围内最大，然后随氯离子浓度的增加而逐渐降低，而无氯离子时几乎不存在pH梯度，直到100 mW时才逐渐增大。醋酸铵或黑曲霉素加K+是pH梯度的分散剂，对膜电位的形成和谷氨酸的吸收几乎没有影响或略有促进作用，而瓦林霉素加K+对膜电位的形成和谷氨酸的吸收都有类似程度的抑制。氟化物、碘化物或硫氰酸盐抑制依赖于ATP和Cl-的谷氨酸摄取，但不影响液泡型HC-ATPase。阴离子通道阻断剂4，4‘-二异硫氰基二苯乙烯-2，2’-二磺酸同样以氯保护的方式抑制谷氨酸的摄取。此外，当存在4 mM Cl-时，MVS发生了依赖于ATP和谷氨酸的酸化。在测试的50多种谷氨酸类似物中，只有少数化合物，包括1-氨基环己烷-反式-1，3-二羧酸，引起了类似的酸化。谷氨酸类似物对谷氨酸摄取的抑制作用与酸化有很好的相关性。这些结果表明：1)膜电位是谷氨酸摄取的主要驱动力，2)氯离子可能通过转运体上的阴离子结合位点(S)调节谷氨酸摄取，3)转运体具有严格的底物特异性。因此，MVS中的囊泡谷氨酸转运体的整体特性与突触囊泡谷氨酸转运体的特性很好地匹配。我们得出结论，囊泡型谷氨酸转运体在内分泌细胞中运作，即使不完全相同，也类似于神经元对应的转运体。
Pinealocytes, endocrine cells that synthesize and secrete melatonin, possess a large number of synaptic-like microvesicles (MVs) containing the L-glutamate transporter (Moriyama, Y., and Yamamoto, A. (1995) FEBS Left., 367, 233-236). In this study, the L-glutamate transporter in MVs isolated from bovine pineal glands was characterized as to its driving force, requirement of anions, and substrate specificity. Upon the addition of ATP, the MVs accumulated L-glutamate. The uptake was significantly dependent on the extravesicular Cl- concentration, being negligible in the absence of Cl- and maximum at 25 mM and decreasing gradually at 20-100 mM. The membrane potential (inside positive) was maximum at 0-10 mM Cl- and then decreased gradually depending on the Cl- concentration, whereas a pH gradient was practically absent without Cl- and increased gradually up to 100 mw Cl-. Ammonium acetate or nigericin plus K+, a dissipator of a pH gradient, had little effect on or was slightly stimulatory toward the uptake, whereas valinomycin plus K+ inhibited both formation of the membrane potential and the glutamate uptake to similar extents. The ATP- and Cl--dependent glutamate uptake was inhibited by fluoride, iodide, or thiocyanate, without vacuolar HC-ATPase being affected. An anion channel blocker, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, similarly inhibited the glutamate uptake in a Cl- protectable manner. Furthermore, ATP- and glutamate-dependent acidification of MVs was observed when 4 mM Cl- was present. Among more than 50 kinds of glutamate analogues tested, only a few compounds, including 1-aminocyclohexane-trans-1,3-dicarboxylic acid, caused similar acidification. A good correlation was observed between the acidification and the inhibition of glutamate uptake by glutamate analogues. These results indicated that 1) the major driving force of the glutamate uptake is the membrane potential, 2) Cl- regulates the glutamate uptake, probably via anion-binding site(s) on the transporter, and 3) the transporter shows strict substrate specificity. Hence, the overall properties of the vesicular glutamate transporter in the MVs well matched those of the synaptic vesicle glutamate transporter. We concluded that the vesicular glutamate transporter, being similar if not identical to the neuronal counterpart, operates in endocrine cells.