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Abstract: FR-PO506

Soluble Klotho Regulates TRPC6 Calcium Signaling via Lipid Rafts and Independently of FGFR-FGF23 Pathway

Session Information

Category: Bone and Mineral Metabolism

  • 401 Bone and Mineral Metabolism: Basic

Authors

  • Xie, Jian, Carver school of Medicine, University of Iowa, Iowa City, Iowa, United States
  • An, Sung Wan, Carver school of Medicine, University of Iowa, Iowa City, Iowa, United States
  • Huang, Chou-Long, Carver school of Medicine, University of Iowa, Iowa City, Iowa, United States
Background

Membranous Klotho interacts with fibroblast growth factor receptor (FGFR) to form coreceptors for FGF23. The ectodomain of membranous klotho is shed (soluble klotho, sKL) and functions as a circulating endocrine or local paracrine factor. sKL protects the heart by inhibiting growth factor-stimulated, PI3K-dependent TRPC6-mediated Ca entry. Lipid rafts are membrane microdomain important in many cellular processes including growth factor signaling and membrane trafficking. We have shown that sKL binds to sialogangliosides of membrane lipid rafts to inhibit cardiac Ca signaling. Regulation of lipid raft formation and function may underlie pleiotropic actions of sKL. A recent X-ray crystal structure of sKL in ternary complex with FGFR and FGF23 suggests that function of sKL is mediated by FGFR and FGF23. The physiological circulating levels of sKL and FGF23 are ~30 pM and 2 pM (50 pg/ml), respectively. Yet many in vitro assays examining the function of sKL, FGF23, and FGFR based on formation of the ternary complex utilize supraphysiological levels of sKL and FGF23. For example, in cell proliferation assay based on sKL-FGF23-FGFR ternary complex, 100 fold higher sKL (3 nM) and 4,000 fold higher FGF23 (200 ng/ml = 4 nM) are commonly used. Here, we examine whether sKL can function independently of FGFR-FGF23.

Methods

Computer modeling of sKL structure with and without FGFR-FGF23. Whole-cell patch-clamp recording of TRPC6 channels in L6 myoblast cell line lacking endogenous FGF receptors.

Results

Computer modeling of sKL structure suggests potential binding sites for sialic acids of gangliosides in the absence of FGFR-FGF23. sKL inhibits TRPC6 channel in L6 cells. We identified amino acid sequence unrelated to sKL but structurally conserved for binding sialic acid known as carbohydrate binding motif (CBM). Purified recombinant CBM inhibits TRPC6 expressed in L6 cells and in HEK293 cells.

Conclusion

Our results support the hypothesis that sKL protects the heart by downregulating TRPC6-mediated calcium signaling in lipid rafts. sKL can exert actions independently of FGFR-FGF23 signaling pathway.

Funding

  • NIDDK Support