Abstract：
Amongst the superfamily of transient receptor potential (TRP) channels, TRPV5 and TRPV6 are specialized members that mediate Ca2+-selective transport across epithelial membranes. Intriguingly, fluorescent fusion proteins of TRPV5 or TRPV6 are hardly discernible within the plasma membrane of living cells. Instead, TRPV6 is mostly found in vesicular membrane compartments, indicating either a rapid degradation or cycling of channel-bearing vesicles between endomembrane compartments and the plasma membrane. In TRPV6-expressing cells, brefeldin A, a toxin that blocks the transit between the endoplasmic reticulum and the Golgi apparatus, caused a drop in [Ca2+]i with a half time in the range of 0.5-1 h. Upon wash-out of the toxin, the [Ca2+]i rose to a steady-state level within 2-3 h. Consistently, the synchronized forward trafficking of TRPV6VL-eGFP after brefeldin A wash-out led to a visible accumulation of the protein within the plasma membrane, as shown by confocal and total internal reflection microscopy. Analysis of the internalization route and differentiation of vesicle populations provided evidence for a clathrin-dependent internalization pathway. Most TRPV6VL-bearing vesicles co-stained with Rab5a, a marker protein for early endosomes. Fewer vesicles were co-localized with Rab7a (late endosomes) or with Rab11 (recycling endosomes). From these data, we propose that the lack of plasma membrane visibility of the channel results from a rapid internalization, which in addition to transcriptional regulation, adds a layer of functional channel regulation to modulate transepithelial Ca2+ transport.
摘要：
在瞬时受体电位（TRP）通道超家族中，TRPV5和TRPV6是介导Ca2+选择性转运穿过上皮膜的专门成员。有趣的是，TRPV5或TRPV6的荧光融合蛋白在活细胞的质膜内几乎不可见。相反，TRPV6主要存在于囊泡膜隔室中，表明内膜隔室和质膜之间的含通道囊泡快速降解或循环。在TRPV6表达细胞中，BrefeldinA,一种阻止内质网和高尔基体之间转运的毒素，在0.5-1小时的范围内引起[Ca2]i的下降。在清除毒素后，[Ca2+]i在2-3小时内升至稳态水平。BrefeldinA洗脱后TRPV6VL-eGFP的同步正向运输导致质膜内蛋白质的可见积累，如共焦和全内反射显微镜所示。对囊泡种群内化途径和分化的分析为网格蛋白依赖性内化途径提供了证据。大多数带有TRPV6VL的囊泡与Rab5a共染色，早期内体的标记蛋白。较少的囊泡与Rab7a（晚期内体）或Rab11（再循环内体）共定位。从这些数据来看,我们认为，缺乏质膜的可见性的通道的结果是一个快速的内在化，除了转录调节，增加了一层功能通道调节以调节跨上皮Ca2转运。
