Real-time detection of the surface delivery of newly synthesized membrane proteins
Real-time detection of the surface delivery of newly synthesized membrane proteins
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DOI：
10.1073/pnas.93.15.7661
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发表时间：
1996-07-23
影响因子：
11.1
通讯作者：
Caplan, MJ
中科院分区：
文献类型：
--
作者：
Andreose, JS;Fumagalli, G;Caplan, MJ
新合成的膜蛋白通过运输囊泡从高尔基复合体运输到细胞表面。利用烟碱型乙酰胆碱受体（AChR）的离子通道特性，真实的实时观察了新合成的AChR蛋白质向培养的肌细胞质膜的组成性传递。采用全细胞电压钳技术，监测了氨甲酰胆碱在新合成的AChR插入质膜后，从20 ℃温度阻断释放时引起的电流波动。我们发现囊泡转移到细胞表面发生在阻断释放后的几分钟内，电信号的时间过程与许多瞬时融合事件一致，尽管有些似乎揭示了融合孔的闪烁，含有AChR的囊泡可以单独融合或作为聚集体融合，细胞内应用鸟苷5 '-[γ-硫代]三磷酸可抑制大多数细胞中AChR的组成性运输。单个胞吐囊泡携带10至300个AChR分子，这表明AChR可能非常密集。
Newly synthesized membrane proteins travel from the Golgi complex to the cell surface in transport vesicles. We have exploited the ion channel properties of the nicotinic acetylcholine receptor (AChR) to observe in real time the constitutive delivery of newly synthesized AChR proteins to the plasma membrane in cultured muscle cells, Whole-cell voltage clamp was employed to monitor the current fluctuations induced by carbamylcholine upon the insertion into the plasma membrane of newly synthesized AChRs, following release from a 20 degrees C temperature block. We find that the transit of vesicles to the cell surface occurs within a few minutes after release of the block, The time course of electrical signals is consistent with many of the fusion events being instantaneous, although some appear to reveal the flickering of a fusion pore, AChR-containing vesicles can fuse individually or as conglomerates, Intracellular application of guanosine 5'-[gamma-thio]triphosphate inhibits the constitutive traffic of AChRs in most cells. Individual exocytotic vesicles carry between 10 and 300 AChR molecules, suggesting that AChRs may be packed extremely densely.