Leunissen Elizabeth H P, Blanchard Maxime G, Sheedfar Fareeba, Lavrijsen Marla, van der Wijst Jenny, Bindels René J M, Hoenderop Joost G J
Department of Physiology, Radboud University Medical Center, Radboud Institute for Molecular Life Sciences, Nijmegen 6500 HB, The Netherlands.
Department of Physiology, Radboud University Medical Center, Radboud Institute for Molecular Life Sciences, Nijmegen 6500 HB, The Netherlands
Glycobiology. 2016 May;26(5):472-81. doi: 10.1093/glycob/cwv172. Epub 2016 Jan 7.
Transcellular Ca(2+)transport in the late distal convoluted tubule and connecting tubule (DCT2/CNT) of the kidney is a finely controlled process mediated by the transient receptor potential vanilloid type 5 (TRPV5) channel. A complex-type-N-glycan bound at the extracellular residue Asn358 of TRPV5 through post-translational glycosylation has been postulated to regulate the activity of TRPV5 channels. Using in vitro Ca(2+)transport assays, immunoblot analysis, immunohistochemistry, patch clamp electrophysiology and total internal reflection fluorescence microscopy, it is demonstrated that the glycosidase β-galactosidase (β-gal), an enzyme that hydrolyzes galactose, stimulates TRPV5 channel activity. However, the activity of the non-glycosylated TRPV(N358Q)mutant was not altered in the presence of β-gal, showing that the stimulation is dependent on the presence of the TRPV5N-glycan. In addition, β-gal was found to stimulate transcellular Ca(2+)transport in isolated mouse primary DCT2/CNT cells. β-gal expression was detected in the apical membrane of the proximal tubules, and the protein was found in mouse urine. In summary, β-gal is present in the pro-urine from where it is thought to stimulate TRPV5 activity.
肾脏远曲小管末端和连接小管（DCT2/CNT）中的跨细胞钙离子转运是一个由瞬时受体电位香草酸亚型5（TRPV5）通道介导的精细调控过程。据推测，通过翻译后糖基化作用，在TRPV5细胞外残基Asn358处结合的复合型N-聚糖可调节TRPV5通道的活性。通过体外钙离子转运分析、免疫印迹分析、免疫组织化学、膜片钳电生理学和全内反射荧光显微镜技术，研究表明，可水解半乳糖的糖苷酶β-半乳糖苷酶（β-gal）能刺激TRPV5通道活性。然而，在β-gal存在的情况下，非糖基化TRPV（N358Q）突变体的活性并未改变，这表明该刺激作用依赖于TRPV5 N-聚糖的存在。此外，研究发现β-gal能刺激分离的小鼠原代DCT2/CNT细胞中的跨细胞钙离子转运。在近端小管的顶端膜中检测到了β-gal的表达，并且在小鼠尿液中发现了该蛋白。总之，β-gal存在于原尿中，据认为它可在原尿中刺激TRPV5活性。
