The epithelial calcium channel TRPV5 is regulated differentially by klotho and sialidase

Abstract

The transient receptor potential vanilloid type 5 (TRPV5) Ca2+ channel facilitates transcellular Ca2+ 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 Ca2+ 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 (TRPV5N358Q) 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 increasesTRPV5activity. 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 klothomediated effect that is dependent on theN-glycan of TRPV5 and a sialidase-mediated stimulation that is lipid raft-dependent and independent of the N-glycan of TRPV5. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.