From the Department of Physiology and.
J Biol Chem. 2013 Oct 11;288(41):29238-46. doi: 10.1074/jbc.M113.473520. Epub 2013 Aug 22.
The transient receptor potential vanilloid type 5 (TRPV5) Ca(2+) channel facilitates transcellular Ca(2+) transport in the distal convoluted tubule (DCT) of the kidney. The channel is glycosylated with a complex type N-glycan and it has been postulated that hydrolysis of the terminal sialic acid(s) stimulate TRPV5 activity. The present study delineates the role of the N-glycan in TRPV5 activity using biochemical assays in Human Embryonic Kidney 293 cells expressing TRPV5, isoelectric focusing and total internal reflection fluorescent microscopy. The anti-aging hormone klotho and other glycosidases stimulate TRPV5-dependent Ca(2+) uptake. Klotho was found to increase the plasma membrane stability of TRPV5, via the TRPV5 N-glycan. Sialidase mimicked this stimulatory action. However, this effect was independent of the N-glycosylation state of TRPV5, since the N-glycosylation mutant (TRPV5(N358Q)) was activated to the same extent. We showed that the increased TRPV5 activity after sialidase treatment is caused by inhibition of lipid raft-mediated internalization. In addition, sialidase modified the N-glycan of transferrin, a model glycoprotein, differently from klotho. Previous studies showed that after klotho treatment, galectin-1 binds the TRPV5 N-glycan and thereby increases TRPV5 activity. However, galectin-3, but not galectin-1, was expressed in the DCT. Furthermore, an increase in TRPV5-mediated Ca(2+) uptake was detected after galectin-3 treatment. In conclusion, two distinct TRPV5 stimulatory mechanisms were demonstrated; a klotho-mediated effect that is dependent on the N-glycan of TRPV5 and a sialidase-mediated stimulation that is lipid raft-dependent and independent of the N-glycan of TRPV5.
瞬时受体电位香草酸亚型 5（TRPV5）钙通道促进肾脏远曲小管（DCT）的细胞间钙转运。该通道与复杂的 N-糖基化聚糖糖基化，并且有人假设末端唾液酸（s）的水解刺激 TRPV5 活性。本研究使用表达 TRPV5 的人胚肾 293 细胞中的生化测定、等电聚焦和全内反射荧光显微镜，阐明了 N-聚糖在 TRPV5 活性中的作用。抗衰老激素 klotho 和其他糖苷酶刺激 TRPV5 依赖性 Ca（2+）摄取。发现 klotho 通过 TRPV5 N-聚糖增加 TRPV5 的质膜稳定性。唾液酸酶模拟了这种刺激作用。然而，这种作用与 TRPV5 的 N-糖基化状态无关，因为 N-糖基化突变体（TRPV5（N358Q））被激活到相同程度。我们表明，唾液酸酶处理后 TRPV5 活性增加是由于抑制脂筏介导的内化。此外，唾液酸酶修饰转铁蛋白（一种模型糖蛋白）的 N-聚糖，与 klotho 不同。先前的研究表明，klotho 处理后，半乳糖凝集素-1 结合 TRPV5 N-聚糖，从而增加 TRPV5 活性。然而，在 DCT 中表达的是半乳糖凝集素-3，而不是半乳糖凝集素-1。此外，在半乳糖凝集素-3 处理后检测到 TRPV5 介导的 Ca（2+）摄取增加。总之，证明了两种不同的 TRPV5 刺激机制；klotho 介导的效应依赖于 TRPV5 的 N-聚糖，以及唾液酸酶介导的刺激，该刺激依赖于脂筏，与 TRPV5 的 N-聚糖无关。
