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. 2014 Dec 26;289(52):35849-57.
doi: 10.1074/jbc.M114.616649. Epub 2014 Nov 5.

Klotho up-regulates renal calcium channel transient receptor potential vanilloid 5 (TRPV5) by intra- and extracellular N-glycosylation-dependent mechanisms

Matthias T F Wolf  1 , Sung-Wan An  2 , Mingzhu Nie  3 , Manjot S Bal  3 , Chou-Long Huang  4
Affiliations

Klotho up-regulates renal calcium channel transient receptor potential vanilloid 5 (TRPV5) by intra- and extracellular N-glycosylation-dependent mechanisms

Matthias T F Wolf et al. J Biol Chem. .

Abstract

The anti-aging protein Klotho is a type 1 membrane protein produced predominantly in the distal convoluted tubule. The ectodomain of Klotho is cleaved and secreted into the urine to regulate several ion channels and transporters. Secreted Klotho (sKL) up-regulates the TRPV5 calcium channel from the cell exterior by removing sialic acids from N-glycan of the channel and inhibiting its endocytosis. Because TRPV5 and Klotho coexpress in the distal convoluted tubule, we investigated whether Klotho regulates TRPV5 action from inside the cell. Whole-cell TRPV5-mediated channel activity was recorded in HEK cells coexpressing TRPV5 and sKL or membranous Klotho (mKL). Transfection of sKL, but not mKL, produced detectable Klotho protein in cell culture media. As for sKL, mKL increased TRPV5 current density. The role of sialidase activity of mKL acting inside is supported by findings that mutations of putative sialidase activity sites in sKL and mKL abrogated the regulation of TRPV5 but that the extracellular application of a sialidase inhibitor prevented the regulation of TRPV5 by sKL only. Mechanistically, coexpression with a dominant-negative dynamin II prevented the regulation of TRPV5 by sKL but not by mKL. In contrast, blocking forward trafficking by brefeldin A prevented the effect with mKL but not with sKL. Therefore, Klotho up-regulates TRPV5 from both the inside and outside of cells. The intracellular action of Klotho is likely due to enhanced forward trafficking of channel proteins, whereas the extracellular action is due to inhibition of endocytosis. Both effects involve putative Klotho sialidase activity. These effects of Klotho may play important roles regarding calcium reabsorption in the kidney.

Keywords: Biotin; Calcium Channel; Electrophysiology; Glycosylation; Intracellular Trafficking; Klotho; Membrane Trafficking; N-glycosylation; Renal Physiology; TRPV5 Channel.

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Figures

FIGURE 1.
FIGURE 1.
Although it is barely detected in the unconcentrated culture medium of transfected cells, membranous Klotho up-regulates TRPV5 whole-cell current similarly as secreted Klotho. A, an abundance of Klotho in unconcentrated media from cells transfected with 400 or 800 ng of sKL or mKL. Note the abundant expression of mKL relative to sKL in cell lysates. B, secreted and membranous Klotho both up-regulate TRPV5 whole-cell current. The bar graph shows TRPV5 current density (picoampere/picofarad (pF)) at −150 mV cotransfected with control empty vector or plasmids for sKL or mKL. Both sKL and mKL increase the TRPV5 whole-cell current compared with cotransfection with the control (n = 5 for each). *, p < 0.05. Typical current traces are shown below each specific study group.
FIGURE 2.
FIGURE 2.
Membranous Klotho increases TRPV5 cell surface abundance. TRPV5 cell surface abundance measured by biotinylation is increased when cotransfected with mKL compared with the control. TRPV5 abundance at the plasma membrane (top) and total lysates (center) were analyzed by immunoblot analysis using antibody against GFP. The antibody did not detect a protein signal in untransfected cells (data not shown). The double bands of TRPV5 reflect unglycosylated and glycosylated protein products. For detection of TRPV5 on the surface and in the lysate, 200 and 10 μg of protein were separated by SDS gel electrophoresis, respectively.
FIGURE 3.
FIGURE 3.
TRPV5 up-regulation by membranous Klotho depends on TRPV5 N-glycosylation. A, in contrast to WT TRPV5, no significant increase of TRPV5 whole-cell current was detected when N-glycan-deficient N358Q TRPV5 was cotransfected with mKL. The bar graph shows the mean ± S.E. (n = 7 for each) of TRPV5 current density at −150 mV. *, p < 0.0001; ns, not significant. pF, picofarad. B, wild-type TRPV5 and N-glycan-deficient N358Q TRPV5 protein densities in total cell lysates do not show any significant difference regarding channel expression with or without mKL cotransfection.
FIGURE 4.
FIGURE 4.
Identification of critical residues of Klotho for up-regulation of TRPV5 via the putative sialidase activity. A, amino acid homology between guinea pig β-glucosidase (gpGD) and the mouse Klotho KL1 and KL2 domains (9). Although glutamate is substituted at the acid-base catalyst position (a-b) by asparagine in the mKL1 domain, glutamate is conserved in the KL2 domain. At the nucleophile (n) position, glutamate is conserved in the mKL1 domain but substituted by alanine (shown) or serine (not shown), respectively. B, mutagenesis of evolutionary conserved amino acids positioned around the acid-base catalyst and nucleophile position in secreted Klotho showed that amino acid residues Glu-416, Asn-417, Asn-690 and Glu-691 were crucial for sKL sialidase activity. The bar graph shows mean ± S.E. (n = 5 for each) of TRPV5 current density at −150 mV. *, p < 0.005; ns, not significant. Typical current traces are shown below each specific study group. C, the lack of TRPV5 stimulation by sKL mutants E416A, N417A, N690A, and E691A is not due to impaired mutant protein expression. Equal volumes of cell culture medium of sKL mutant and wild-type transfected HEK cells were analyzed by immunoblot. Mutant sKL was either secreted to the same degree (no significant difference between WT and A416A or N690A) or even more (N417A and E691A) compared with WT sKL. Average results (mean ± S.E.) from four experiments are shown. The abundance of WT and mutant KL proteins in culture medium was quantified by densitometry (area under the curve). Representative samples of mutant and corresponding wild-type sKL are shown in the bottom panel. The secreted KL mutants E416A and N417A were transfected in one experiment, and sKL N690A and E691A were transfected in another experiment, which resulted in two different correlating wild-type KL bands as a positive control. The density of protein abundance was normalized to that in the wild-type KL group (given value of 1). *, p < 0.05; #, p < 0.005; ns, not significant.
FIGURE 5.
FIGURE 5.
Membranous KL requires sialidase activity for TRPV5 up-regulation. A, we chose the Klotho E416, N417, N690, and E691 residues as crucial amino acid residues for sKL sialidase activity and performed site-directed mutagenesis in membranous KL. In contrast to wild-type membranous KL, the E416A, N417A, N690A, and E691A mutants of membranous KL did not increase TRPV5 current density (n = 6 for each). *, p < 0.0005; ns, not significant. pF, picofarad. B, TRPV5 protein density in total cell lysate is shown after cotransfection with either the control (pEF), wild-type mKL, or the E416A, N417A, N690A, or E691A mKL mutants. No significant difference regarding TRPV5 channel expression was detected. TRPV5 cotransfected with mKL mutants was at least as well expressed in total cell lysate as when cotransfected with mKL wild-type (mKL).
FIGURE 6.
FIGURE 6.
The extracellular sialidase inhibitor DANA prevents TRPV5 up-regulation by secreted Klotho but not TRPV5 up-regulation by membranous Klotho. Application of DANA to the cell culture medium had no effect on the TRPV5 whole-cell current of HEK cells cotransfected with the vector (Vec) control or mKL but completely abolished TRPV5 up-regulation by sKL. The bar graph shows mean ± S.E. (n = 6 for each) of TRPV5 current density at −150 mV. *, p < 0.005 compared with corresponding vector control-transfected HEK cells; #, p < 0.001; ns, not significant. Typical current traces are shown below each specific study group.
FIGURE 7.
FIGURE 7.
Up-regulation of TRPV5 by membranous Klotho is not affected by impaired dynamin-II dependent endocytosis but is neutralized by impaired forward trafficking. A, HEK cells were cotransfected with TRPV5, sKL, or mKL and with either wild-type (WT-Dyn2) or dominant-negative (K44A) dynamin II (DN-Dyn2). The bar graph shows mean ± S.E. (n = 6 for each) of TRPV5 current density at −150 mV. *, p < 0.0005 compared with vector + WT-Dyn2; #, p < 0.001; ns, not significant. Typical current traces are shown below each specific study group. B, TRPV5 protein density in total cell lysate is shown after cotransfection with control vector, wild-type sKL, or mKL with either WT-Dyn2 or DN-Dyn2. No significant difference regarding TRPV5 channel expression was detected between cells cotransfected with sKL or mKL. C, HEK cells were cotransfected with TRPV5 and with sKL or mKL. Cells were then treated with either dimethyl sulfoxide (DMSO, vehicle) or BFA (38). The bar graph shows mean ± S.E. (n = 5 for each) of TRPV5 current density at −150 mV. *, p < 0.0005 compared with dimethyl sulfoxide-treated, vector control-transfected HEK cells; #, p < 0.001 compared with BFA-treated vector (Vec) control; ns, not significant. Typical current traces are shown below each specific study group.
FIGURE 8.
FIGURE 8.
The effect of Klotho on TRPV5 is independent of FGF23. A, coexpression with cDNA coding for sKL or mKL (1000 ng each) both increased TRPV5 current density compared with vector in L6 cells lacking endogenous FGFRs. There was a trend toward higher TRPV5 current density with mKL, but it was not statistically significant. B, extracellular application of DANA completely abolished the increase of TRPV5 current density by coexpression with sKL but not with mKL. C, coexpression with cDNA coding for sKL or mKL (2000 ng each) both increased TRPV5 current density in L6 cells lacking endogenous FGFRs. The increase by mKL is greater than that by sKL. D, extracellular application of DANA completely abolished the increase of TRPV5 current density by coexpression with sKL (2000 ng cotransfected), and slightly (but not statistically significantly) diminished the increase by mKL (2000 ng cotransfected). The bar graph shows mean ± S.E. (n = 6 for each) of TRPV5 current density at indicated mV. *, p < 0.01 compared with vector; #, p < 0.01 between groups; pF, picofarad.
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