TY - JOUR
T1 - The epithelial calcium channel TRPV5 is regulated differentially by klotho and sialidase
AU - Leunissen, Elizabeth H P
AU - Nair, Anil V
AU - Büll, Christian
AU - Lefeber, Dirk J
AU - van Delft, Floris L
AU - Bindels, René J M
AU - Hoenderop, Joost G J
PY - 2013/10/11
Y1 - 2013/10/11
N2 - 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.
AB - 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.
KW - Animals
KW - Blotting, Western
KW - Calcium/metabolism
KW - Calcium Channels/genetics
KW - Cell Membrane/drug effects
KW - Epithelial Cells/drug effects
KW - Galectin 3/genetics
KW - Glucuronidase/genetics
KW - Glycoside Hydrolases/genetics
KW - Green Fluorescent Proteins/genetics
KW - HEK293 Cells
KW - Humans
KW - Membrane Microdomains/drug effects
KW - Membrane Potentials/drug effects
KW - Mice
KW - Microscopy, Fluorescence
KW - Neuraminidase/genetics
KW - Patch-Clamp Techniques
KW - Polysaccharides/metabolism
KW - Recombinant Proteins/pharmacology
KW - TRPV Cation Channels/genetics
U2 - 10.1074/jbc.m113.473520
DO - 10.1074/jbc.m113.473520
M3 - Journal article
C2 - 23970553
SN - 0021-9258
VL - 288
SP - 29238
EP - 29246
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 41
ER -