Cidon S, Sihra T S
Laboratory of Molecular and Cellular Neuroscience, Rockefeller University, New York, New York 10021-6399.
J Biol Chem. 1989 May 15;264(14):8281-8.
Synaptic vesicles contain a H+-ATPase that generates a proton electrochemical gradient (delta mu H+) required for the uptake of neurotransmitters into the organelles. In this study, the synaptic vesicle H+-ATPase was examined for structural and functional similarities with other identified ATPases that generate a delta mu H+ across membranes. The synaptic vesicle H+-ATPase displayed immunological similarity with the 115-, 72-, and 39-kDa subunits of a vacuolar-type H+-ATPase purified from chromaffin granules. Functionally, the ATP-dependent H+ pumping across synaptic vesicles and ATP hydrolysis were sensitive to the sulfhydryl-modifying reagents, N-ethylmaleimide and 4-chloro-7-nitrobenz-2-oxa-1,3-diazole, at concentrations known to affect vacuolar-type H+-ATPases. In addition, as with vacuolar-type H+-ATPases, the presence of NO3-, SO4(2-), or F- inhibited the generation of a delta mu H+, but addition of vanadate or oligomycin had no effect. The delta mu H+ is a function of the pH gradient (delta pH) and membrane potential (delta psi sv) across the synaptic vesicle. Acidification (delta pH) of the synaptic vesicle interior was enhanced in the presence of permeant anions, such as Cl-, or the K+ ionophore, valinomycin. In the absence of permeant anions, the H+-ATPase generated a delta psi sv that effected the transport of L-glutamate into the synaptic vesicles. Dissipation of delta psi sv by incubation with increased external Cl- or nigericin resulted in the abolition of glutamate uptake, despite the continued maintenance of a delta mu H+ across the synaptic vesicle as a substantial delta pH. The results suggest that the synaptic vesicle H+-ATPase is of a vacuolar type and energizes the uptake of anionic glutamate by virtue of the delta psi sv component of the delta mu H+ it generates.
突触囊泡含有一种H⁺-ATP酶，该酶能产生质子电化学梯度（ΔμH⁺），这是神经递质摄取到细胞器中所必需的。在本研究中，对突触囊泡H⁺-ATP酶与其他已鉴定的能跨膜产生ΔμH⁺的ATP酶进行了结构和功能相似性检测。突触囊泡H⁺-ATP酶与从嗜铬颗粒中纯化的液泡型H⁺-ATP酶的115 kDa、72 kDa和39 kDa亚基表现出免疫相似性。在功能上，跨突触囊泡的ATP依赖性H⁺泵浦和ATP水解对巯基修饰试剂N-乙基马来酰亚胺和4-氯-7-硝基苯并-2-恶唑-1,3-二氮杂苯敏感，其浓度已知会影响液泡型H⁺-ATP酶。此外，与液泡型H⁺-ATP酶一样，NO₃⁻、SO₄²⁻或F⁻的存在会抑制ΔμH⁺的产生，但添加钒酸盐或寡霉素则没有影响。ΔμH⁺是跨突触囊泡的pH梯度（ΔpH）和膜电位（Δψsv）的函数。在存在渗透性阴离子（如Cl⁻）或K⁺离子载体缬氨霉素的情况下，突触囊泡内部的酸化（ΔpH）增强。在没有渗透性阴离子的情况下，H⁺-ATP酶产生了一个Δψsv，它影响L-谷氨酸转运到突触囊泡中。通过与增加的外部Cl⁻或尼日利亚菌素孵育来消散Δψsv，尽管跨突触囊泡作为一个显著的ΔpH持续维持着ΔμH⁺，但谷氨酸摄取仍被消除。结果表明，突触囊泡H⁺-ATP酶属于液泡型，并且通过它所产生的ΔμH⁺的Δψsv成分来为阴离子型谷氨酸的摄取提供能量。
