Chloride Channel-3 (ClC-3) Modifies the Trafficking of Leucine-Rich Repeat-Containing 8A (LRRC8A) Anion Channels.
Chloride Channel-3 (ClC-3) Modifies the Trafficking of Leucine-Rich Repeat-Containing 8A (LRRC8A) Anion Channels.
复制标题
DOI：
10.1007/s00232-022-00271-9
复制
发表时间：
2023-04
期刊：
影响因子：
--
通讯作者：
中科院分区：
文献类型：
--
作者：
氯离子通道-3（ClC-3）Cl−/H+反向转运蛋白和富含亮氨酸重复序列的8（LRRC 8）家族阴离子通道都与容量调节阴离子电流（VRAC）相关。VRAC通常在C1 C-3无效细胞中改变，但在LRRC 8A无效细胞中不存在。为了探索ClC-3、LRRC 8A和VRAC之间的关系，我们使用多模式显微镜在人上皮肾（HEK 293）细胞中定位标记蛋白。ClC-3-GFP的表达诱导了在分界膜中具有ClC-3的大多泡体（MVB）。LRRC 8A-RFP定位于质膜和小的细胞质囊泡。共表达证明了与早期内体标记Rab 5A相关的小的、高度移动的胞质囊泡中的共定位。然而，大多数小的LRRC 8A阳性囊泡被限制在界定膜中具有丰富的C1 C-3的大MVB内。显性负性（S34 A）Rab 5A防止ClC-3过表达产生扩大的MVB，而组成型活性（Q79 L）Rab 5A增强该表型。因此，C1 C-3和LRRC 8A被一起内吞，但在Rab 5A MVB中独立分选。随后，LRRC 8A标记的囊泡被分选为Rab 27 A和B外泌体区室标记物标记的MVB，但不分选为Rab 11再循环内体。VRAC电流在C1 C-3无效HEK 293细胞中显著更大。这项工作证明了LRRC 8A运输对C1 C-3的依赖性，这可以解释C1 C-3和VRAC之间的关联。
Chloride channel-3 (ClC-3) Cl−/H+ antiporters and leucine-rich repeat-containing 8 (LRRC8) family anion channels have both been associated with volume-regulated anion currents (VRACs). VRACs are often altered in ClC-3 null cells but are absent in LRRC8A null cells. To explore the relationship between ClC-3, LRRC8A and VRAC we localized tagged proteins in human epithelial kidney (HEK293) cells using multimodal microscopy. Expression of ClC-3-GFP induced large multivesicular bodies (MVBs) with ClC-3 in the delimiting membrane. LRRC8A-RFP localized to the plasma membrane and to small cytoplasmic vesicles. Co-expression demonstrated co-localization in small, highly mobile cytoplasmic vesicles that associated with the early endosomal marker Rab5A. However, most of the small LRRC8A-positive vesicles were constrained within large MVBs with abundant ClC-3 in the delimiting membrane. Dominant negative (S34A) Rab5A prevented ClC-3 overexpression from creating enlarged MVBs, while constitutively active (Q79L) Rab5A enhanced this phenotype. Thus, ClC-3 and LRRC8A are endocytosed together but independently sorted in Rab5A MVBs. Subsequently, LRRC8A-labelled vesicles were sorted to MVBs labelled by Rab27A and B exosomal compartment markers, but not to Rab11 recycling endosomes. VRAC currents were significantly larger in ClC-3 null HEK293 cells. This work demonstrates dependence of LRRC8A trafficking on ClC-3 which may explain the association between ClC-3 and VRACs.
登录
查看更多内容
DOI：
10.1007/978-1-4939-2569-8_1
发表时间：
2015
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
--
作者：
Li G;Marlin MC
通讯作者：
Marlin MC
影响因子：
7.3
作者：
Dai, YP;Bongalon, S;Yamboliev, IA
通讯作者：
Yamboliev, IA
影响因子：
64.8
作者：
Duan, D;Winter, C;Horowitz, B
通讯作者：
Horowitz, B
影响因子：
4.8
作者：
Cuddapah, Vishnu Anand;Sontheimer, Harald
通讯作者：
Sontheimer, Harald
影响因子：
5.5
作者：
Arreola, J;Begenisich, T;Melvin, JE
通讯作者：
Melvin, JE