Leroy C, Meunier F M, Lesbats B, Israël M
Département de Neurochimie, C.N.R.S., Gif-sur-Yvette, France.
Gen Pharmacol. 1994 Mar;25(2):245-55. doi: 10.1016/0306-3623(94)90050-7.
The mediatophore is a presynaptic oligomeric protein purified from the presynaptic plasma membrane of Torpedo synaptosomes on the basis of its ability to mediate a calcium-dependent acetylcholine release when solubilized and reconstituted into proteoliposomes. We investigated the ACh translocating activity of the 15 kDa proteolipid subunit of the mediatophore when expressed in Xenopus oocytes and reconstituted into proteoliposomes loaded with ACh. 1. A calcium-dependent ACh translocation was observed when oocytes were injected with polyadenylated mRNAs extracted from the electric lobe of the Torpedo brain or with an in vitro transcribed RNA encoding the 15 kDa subunit. 2. No release response was obtained when oocytes were non-injected or injected with Torpedo liver mRNAs. 3. This ACh translocation mechanism showed calcium-dependent activation and desensitisation and was inhibited by cetiedil, sharing these properties with the release of ACh observed at the synapse. 4. The ACh translocating activity of an N terminal deleted mediatophore 15 kDa subunit was strongly reduced and the deleted proteolipid appeared less sensitive to the action of cetiedil (alpha-cyclohexyl-alpha-(3-thienyl)-acetate of perhydroazepinyl-alpha-ethyl citrate monohydrate). 5. A significant ACh release response was observed when the 15 kDa proteolipid of the H(+)-ATPase from bovine chromaffin granules was tested. 6. These results show that this ACh translocating activity could be induced in the oocyte membranes by the expression of the 15 kDa subunit alone.
介质载体是一种从电鳐突触体的突触前质膜中纯化出来的突触前寡聚蛋白，其依据在于当溶解并重新组装到蛋白脂质体中时，它能够介导钙依赖性乙酰胆碱释放。我们研究了介质载体15 kDa蛋白脂质亚基在非洲爪蟾卵母细胞中表达并重新组装到装载有乙酰胆碱的蛋白脂质体中时的乙酰胆碱转运活性。1. 当向卵母细胞注射从电鳐脑电叶中提取的聚腺苷酸化mRNA或编码15 kDa亚基的体外转录RNA时，观察到了钙依赖性乙酰胆碱转运。2. 当卵母细胞未注射或注射电鳐肝脏mRNA时，未获得释放反应。3. 这种乙酰胆碱转运机制表现出钙依赖性激活和脱敏作用，并被西替地尔抑制，与在突触处观察到的乙酰胆碱释放具有这些共同特性。4. N端缺失的介质载体15 kDa亚基的乙酰胆碱转运活性显著降低，且缺失的蛋白脂质对西替地尔（全氢氮杂环庚基-α-乙基柠檬酸单水合物的α-环己基-α-(3-噻吩基)-乙酸）的作用似乎不太敏感。5. 当测试来自牛嗜铬颗粒的H(+)-ATP酶的15 kDa蛋白脂质时，观察到了显著的乙酰胆碱释放反应。6. 这些结果表明，仅通过15 kDa亚基的表达就可以在卵母细胞膜中诱导这种乙酰胆碱转运活性。
