Klotho and bacterial sialidase increase surface abundance of TRPV5. (A) (Upper) Klotho increases whole-cell current density of WT TRPV5 but not of N358Q mutant. HEK cells were transiently transfected with GFP-tagged WT or mutant TRPV5. Twenty-four hours after transfection, cells were incubated with purified KLe for 16–24 h before whole-cell recordings. Whole-cell current density (currents normalized to the cell surface area) was measured by voltage steps from −150 to +100 mV. Voltage-current (I–V) relation curves show characteristic inwardly rectifying currents in TRPV5-transfected cells treated with KLe (+KLe; 100 pM) or without KLe (control). No TRPV5 currents were detected in mock-transfected cells (16). (Lower) Current density (nA/pF; at −100 mV) is shown. *, P < 0.05 vs. vehicle (no KLe; open bar). NS, not significant. (B) Effect of bacterial sialidase on TRPV5. The effect of sialidase (Siad) is not additive to that of KLe (200 pM). Current density (nA/pF; at −100 mV) relative to vehicle (no KLe or Siad; open bar, designated 1) is shown. *, P < 0.05 vs. vehicle. NS, not significant. (C) Effect of DANA on Klotho-induced increase of TRPV5. DANA (0.1 μM) was added to cells simultaneously with KLe (200 pM). *, P < 0.05 vs. vehicle (no KLe or DANA). (D) Effects of KLe and bacterial sialidase on surface abundance of TRPV5. Gly indicates glycosylated TRPV5. (E) Mutations of the putative active site abolished the effect of Klotho on TRPV5. Cells were incubated with purified Klotho (200 pM) or conditioned media (Med) harvested from control HEK cells or cells expressing the extracellular domain of WT Klotho or mutant Klotho carrying NEE → AAA triple mutations (200 pM each). *, P < 0.05 vs. control (CTRL). (F) Effect of medium pH on regulation of TRPV5 by Klotho. HEK cells expressing GFP-TRPV5 were incubated with vehicle or KLe (at indicated concentrations) in a physiological salt solution at pH 6.4 or 7.4 for 15 min. See Fig. 2C for effectiveness of short incubation with Klotho. After extensive washing, cells were further incubated in the cell culture medium (at pH 7.4) for 16 h before recording. The short incubation in acidic pH avoids untoward effects on cells. *, P < 0.05 vs. vehicle (−KLe; open bar).

Galectin-1 is critical for the increase of surface abundance of channels by Klotho. (A) Typical complex-type tetra-antennary N-glycan present in mammalian cell surface glycoproteins (27, 37–40). The Golgi enzyme GnT-V catalyzes the addition of GlcNAc to the α6-mannose, initiating the GlcNAcβ(1,6) branch. The polymeric form of LacNAc_n (n > 1) is frequently present in the GlcNAcβ(1,6) branch. See Fig. 4E legend for further details. (B) Antibody against galectin-1 (GLTN-1), but not against galectin-8 (GLTN-8), prevents the increase of TRPV5 current by Klotho. Transfected cells were incubated with KLe for 30 min, washed off KLe, and incubated with the indicated antibody (15 nM) in a KLe-free medium for 16–24 h before recording. (C) LacNAc (LN), α2,3-sialylated LacNAc (3SLN), but not α2,6-sialylated LacNAc (6SLN), prevents the increase of TRPV5 current by Klotho. Cells were incubated with KLe for 30 min, washed off KLe, and incubated with the indicated compounds (10 mM) (37, 38) in a KLe-free medium for 16–24 h before recording. (D) Galectin-1 (GLTN-1) coimmunoprecipitates with TRPV5 after treatment by Klotho. Cells were transfected with WT GFP-TRPV5 or N358Q mutant, incubated with or without Klotho, and cross-linked by DTSSP before lysis and immunoprecipitated by anti-GFP antibodies. Cross-linked proteins were reduced before separation by gel electrophoresis. (E) Terminal galactose residues are required for the increase of TRPV5 current by Klotho. Cells were incubated with KLe and/or galactosidase (GalDase; 0.5 units/ml) for 1 h, washed off, and further incubated in a KLe-free medium for 16–24 h before recording. In the experiment labeled Before KL, cells were treated with GalDase for 1 h, washed off with GalDase, incubated with KLe for 1 h, washed off withKLe, and incubated in a KLe-free medium for 16–24 h before recording. *, P < 0.05 vs. vehicle control (open bars).

Treatment by Klotho prevents binding of SNA to TRPV5. (A and B) Control CHO cells were incubated with biotinylated MAA (A) or SNA (B), followed by Alexa 594-labeled streptavidin. (C–F) CHO cells were transfected with GFP-TRPV5 and ST6Gal-1 and treated with Klotho (500 pM) (E and F) or without Klotho (C and D) for 1 h before staining by SNA. In each experimental condition, fluorescent images were acquired by gating for GFP fluorescence (C and E) and Alexa 594 fluorescence (D and F) to detect transfected cells and labeling by SNA, respectively. White arrow in F indicates that cells with relatively less TRPV5 expression are less sensitive to Klotho, which is consistent with the notion that Klotho is specific for α2,6-sialic acids from glycan chains of TRPV5, but not from the majority resident surface membrane proteins in CHO cells (see I–M). (G and H) CHO cells were cotransfected with GFP and ST3Gal-1 and stained by SNA. (I–M) CHO cells were cotransfected with GFP and ST6Gal-1 and treated with (K and M) or without Klotho (I and J) for 1 h before staining by SNA.

Klotho decreases dynamin-dependent internalization of TRPV5. (A) Effect of dominant-negative (DN) dynamin on TRPV5 currents with or without treatment by Klotho (KLe). WT dynamin was used as control. *, P < 0.05 vs. vehicle (−KLe; open bar). NS, not significant. (B) Time course of increase in TRPV5 currents with continuous Klotho incubation. TRPV5-transfected cells were incubated with KLe (200 pM) for 0.5, 3, 16, and 24 h. Currents were recorded at the end of each period of incubation (i.e., at 0.5, 3, 16, or 24 h, respectively). (C) Time course of increase in TRPV5 currents with brief incubation of Klotho. TRPV5-transfected cells were first incubated with KLe (200 pM) for 15, 30, or 60 min, washed extensively to remove KLe, and further incubated in a KLe-free medium for 16–24 h. Currents were recorded at the end of 16–24 h of incubation in the KLe-free medium. For comparison, some cells were incubated with KLe continuously for 16 h and recorded at end of 16 h of incubation (Cont. KLe 16 h).

Sialyltransferase ST6Gal-1 is responsible for the synthesis of sialic acid substrate for Klotho. (A) Knockdown of ST6s, but not ST3s and ST8s, in HEK cells prevents the increase of TRPV5 current by Klotho. Cells were transfected with GFP-TRPV5 mixed with control oligonucleotide or pooled RNAi oligonucleotides for ST3s, ST6s, or ST8s. (B) Knockdown of ST6Gal-1, but not ST6Gal-2 and other ST6s, in HEK cells prevents the increase of TRPV5 current by Klotho. Cells were transfected with GFP-TRPV5 mixed with control oligonucleotide or RNAi oligonucleotides for ST6Gal-1, ST6Gal-2, or pooled RNAi oligonucleotides for other ST6s. (C) Klotho increases TRPV5 currents in ST6Gal-1-transfected but not control or ST3Gal-1-transfected CHO cells. (D) Anti-galectin-1 antibody prevents the increase of TRPV5 current by Klotho in ST6Gal-1-transfected CHO cells. (E) Klotho increased TRPV5 current density in Lec4 cells expressing both ST6Gal-1 and GnT-V, but not in Lec4 cells expressing only ST6Gal-1 or only GnT-V. In A–E, *, P < 0.05 vs. vehicle control (−KLe; open bar). (F) Effect of bacterial sialidase on TRPV5 in CHO cells. Cells were cotransfected with GFP-TRPV5 plus ST6Gal-1 or an empty vector and incubated with bacterial sialidase (0.5 units/ml) for 16 h. *, P < 0.05 vs. vehicle (−sialidase; open bar).

Model for increase in cell-surface abundance of TRPV5 by Klotho. Klotho removes terminal sialic acid from the GlcNAcβ(1,6) branch of (tri-antenary or) tetra-antenary N-glycan of TRPV5. Removal of sialic acids exposes underlying LacNAc, a ligand for galectin-1. Binding to galectin-1 lattice at the extracellular surface leads to accumulation of functional TRPV5 on the plasma membrane.