Light inactivation of water transport and protein-protein interactions of aquaporin-Killer Red chimeras.
Light inactivation of water transport and protein-protein interactions of aquaporin-Killer Red chimeras.
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DOI：
10.1085/jgp.201110712
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发表时间：
2012-01
期刊：
影响因子：
--
通讯作者：
Verkman AS
中科院分区：
文献类型：
--
作者：
Baumgart F;Rossi A;Verkman AS
水通道蛋白（AQPs）具有广泛的细胞和器官功能；然而，目前还没有无毒的AQP水转运抑制剂。在这里，我们应用了发色团辅助光失活（CALI）来抑制AQP1和两个AQP4亚型（M1和M23）的水渗透性，其中一个（M23）在细胞膜上形成聚集体。含有杀手红（KR）和AQPs的嵌合体具有不同长度的连接体。表达KR/AQP嵌合体的细胞通过渗透膨胀诱导的细胞质氯化物稀释来测量渗透水渗透性，使用基因编码的氯化物感应荧光蛋白检测。KR-AQP1红色荧光被宽视场荧光显微镜快速漂白（每秒约10%）。KR漂白后，连接最短的嵌合体KR- aqp1的透水性降低了80%。值得注意的是，cali诱导的AQP4-KR水渗透性降低对形成聚集的M23亚型的效率大约是其两倍；这可能是分子间CALI，通过天然凝胶电泳证实了共表达M23-AQP4- kr和myc标记的M23-AQP4的细胞。CALI还破坏了AQP4与针对AQP4细胞外表位的视神经脊髓炎自身抗体的相互作用。因此，CALI可以快速、有空间针对性和不可逆地降低AQP的水渗透性和活细胞中的相互作用。我们的数据也支持CALI在研究蛋白-蛋白相互作用以及其他膜转运体和受体方面的效用。
Aquaporins (AQPs) have a broad range of cellular and organ functions; however, nontoxic inhibitors of AQP water transport are not available. Here, we applied chromophore-assisted light inactivation (CALI) to inhibit the water permeability of AQP1, and of two AQP4 isoforms (M1 and M23), one of which (M23) forms aggregates at the cell plasma membrane. Chimeras containing Killer Red (KR) and AQPs were generated with linkers of different lengths. Osmotic water permeability of cells expressing KR/AQP chimeras was measured from osmotic swelling–induced dilution of cytoplasmic chloride, which was detected using a genetically encoded chloride-sensing fluorescent protein. KR-AQP1 red fluorescence was bleached rapidly (∼10% per second) by wide-field epifluorescence microscopy. After KR bleaching, KR-AQP1 water permeability was reduced by up to 80% for the chimera with the shortest linker. Remarkably, CALI-induced reduction in AQP4-KR water permeability was approximately twice as efficient for the aggregate-forming M23 isoform; this suggests intermolecular CALI, which was confirmed by native gel electrophoresis on cells coexpressing M23-AQP4-KR and myc-tagged M23-AQP4. CALI also disrupted the interaction of AQP4 with a neuromyelitis optica autoantibody directed against an extracellular epitope on AQP4. CALI thus permits rapid, spatially targeted and irreversible reduction in AQP water permeability and interactions in live cells. Our data also support the utility of CALI to study protein–protein interactions as well as other membrane transporters and receptors.
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影响因子：
7.4
作者：
McLean, Mark A.;Rajfur, Zenon;Chen, Zaozao;Humphrey, David;Yang, Bing;Sligar, Stephen G.;Jacobson, Ken
通讯作者：
Jacobson, Ken
DOI：
10.1073/pnas.0902725106
发表时间：
2009-05-05
影响因子：
11.1
作者：
Ho, Joseph D.;Yeh, Ronald;Stroud, Robert M.
通讯作者：
Stroud, Robert M.
影响因子：
3.5
作者：
Huber, Vincent J.;Tsujita, Mika;Nakada, Tsutomu
通讯作者：
Nakada, Tsutomu
影响因子：
21.3
作者：
Rajfur, Z;Roy, P;Jacobson, K
通讯作者：
Jacobson, K
DOI：
10.1073/pnas.91.7.2659
发表时间：
1994-03-29
影响因子：
11.1
作者：
LIAO, JC;ROIDER, J;JAY, DG
通讯作者：
JAY, DG