Lauf Undine, Giepmans Ben N G, Lopez Patricia, Braconnot Sebastien, Chen Shu-Chih, Falk Matthias M
Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Proc Natl Acad Sci U S A. 2002 Aug 6;99(16):10446-51. doi: 10.1073/pnas.162055899. Epub 2002 Jul 29.
Certain membrane channels including acetylcholine receptors, gap junction (GJ) channels, and aquaporins arrange into large clusters in the plasma membrane (PM). However, how these channels are recruited to the clusters is unknown. To address this question, we have investigated delivery of GJ channel subunits (connexons) assembled from green fluorescent protein (GFP)-tagged connexin 43 (Cx43) to the PM and GJs in living cells. Fluorescence-photobleaching of distinct areas of Cx43-GFP GJs demonstrated that newly synthesized channels were accrued to the outer margins of channel clusters. Time-lapse microscopy further revealed that connexons were delivered in vesicular carriers traveling along microtubules from the Golgi to the PM. Routing and insertion of connexons occurred predominantly into the nonjunctional PM. These PM connexons can move laterally as shown by photo-bleaching and thus, can reach the margins of channel clusters. There, the apposing PMs are close enough to allow connexons to dock into complete GJ channels. When connexon delivery to the PM was inhibited by brefeldin A, or nocodazole pretreatment, the PM pool initially enabled connexon accrual to the clusters but further accrual was inhibited upon depletion. Taken together, our results indicate that GJ channel clusters grow by accretion at their outer margins from connexon subunits that were delivered to the nonjunctional PM, and explain how connexons in the PM can function in intra-/extracellular signaling before GJ channel formation and direct cell-cell communication.
某些膜通道，包括乙酰胆碱受体、间隙连接（GJ）通道和水通道蛋白，在质膜（PM）中排列成大的簇。然而，这些通道如何被招募到簇中尚不清楚。为了解决这个问题，我们研究了由绿色荧光蛋白（GFP）标记的连接蛋白43（Cx43）组装而成的GJ通道亚基（连接子）向活细胞中的质膜和间隙连接的转运。对Cx43-GFP间隙连接的不同区域进行荧光光漂白表明，新合成的通道聚集在通道簇的外缘。延时显微镜进一步显示，连接子通过沿着微管从高尔基体向质膜移动的囊泡载体进行转运。连接子的路由和插入主要发生在非连接质膜中。如光漂白所示，这些质膜连接子可以横向移动，因此可以到达通道簇的边缘。在那里，相对的质膜足够接近，使得连接子能够对接形成完整的GJ通道。当用布雷菲德菌素A或诺考达唑预处理抑制连接子向质膜的转运时，质膜池最初使连接子能够聚集到簇中，但在耗尽后进一步的聚集受到抑制。综上所述，我们的结果表明，GJ通道簇通过从转运到非连接质膜的连接子亚基在其外缘的积累而生长，并解释了质膜中的连接子在GJ通道形成和直接细胞间通讯之前如何在细胞内/外信号传导中发挥作用。
