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

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 -