8.A.49 The Klotho Auxiliary Protein (Klotho) Family The Klotho protein in mammals activates a number of transporters, but also affects aging and aging-related conditions such as neurodegeneration and muscle wasting. It is a large membrane-embeded protein with two TMSs, one N-terminal and one C-terminal. Its extracellular domain has β-glucuronidase activity with two domains of the BglB (Glyco_hydro_1) superfamily which comprise virtually all of the extracellular domain between the two TMSs, and these may play a role in enhancing or inhibiting various transport activities. It activates CreaT (SLC6A8) by stabilizing the carrier in the plasma membrane (Almilaji et al. 2014). The direct role of alphaKlotho and FGF23 in the maintenance of phosphate homeostasis is partly mediated by modulation of type II Na⁺-dependent phosphate co-transporters in target organs. αKlotho and FGF23 are principal phosphotropic hormones (Hu et al. 2018). Soluble αKlotho downregulates Orai1-mediated store-operated Ca²⁺ entry via PI3K-dependent signaling (Kim et al. 2021). Klotho inhibits H₂O₂-induced oxidative stress and apoptosis in periodontal ligament stem cells by regulating UCP2 (TC# 2.A.29.3.4) expression (Zhu et al. 2021). Klotho also regulates diverse calcium and potassium ion channels as well as several carriers including the Na⁺-coupled excitatory amino acid transporters EAAT1, 2, 3 and 4 which are up-regulated (Warsi et al. 2015), the Na⁺-coupled phosphate cotransporters, NaP_i-IIa and NaP_i-IIb, and the Na⁺/K⁺-ATPase. All these cellular transport regulations contribute to the aging suppressor role of Klotho (Sopjani et al. 2014). Klotho expressed in the proximal tubules has a defined but limited role in renal phosphate handling in vivo (Ide et al. 2016). The expression of Ca²⁺ channels, including Orai3, but not Orai1, Orai2, TRPV5 and TRPV6 was reduced in KL-silenced as compared to control cells (Xuan and Hai 2018). Klotho in humans is encoded by the hKL gene. This aging suppressor protein is predominantly expressed in the distal convoluted tubule of the kidney, parathyroid glands, and choroid plexus of the brain. The Klotho protein exists in both a full-length membrane form and a soluble secreted form. The extracellular domain can be enzymatically cleaved off and released into the systemic circulation where it functions as both a beta-glucuronidase and a hormone. Soluble Klotho (sKlotho) is present in biological fluids including blood, urine and cerebrospinal fluids. Klotho deficiency leads to multi-organ failure accompanied by early appearance of multiple age-related disorders and early death, whereas overexpression results in the opposite effects. Klotho regulates plasma membrane transport either indirectly through inhibiting calcitriol (1,25(OH)2D3) formation (or another mechanism), or by directly binding to and affecting transporter proteins. As noted above, these include ion channels, cellular carriers, and the Na⁺/K⁺-ATPase (Sopjani and Dërmaku-Sopjani 2016). alpha-Klotho, a type 1 transmembrane protein, exhibits aging suppression function. The large amino-terminal extracellular domain of α-klotho is shed as soluble klotho (sKlotho) and functions as a circulating cardioprotective hormone as noted above. Diacylglycerol (DAG)-activated calcium-permeable TRPC6 channels play a critical role in stress-induced cardiac remodeling. DAG activates TRPC6 by acting directly on the channel to increase its activity and by stimulation of channel exocytosis. sKlotho protects the heart by inhibiting DAG stimulation of TRPC6 exocytosis. Using a compound that directly activates TRPC6 without affecting channel exocytosis, Xie et al. 2020 showed that sKlotho selectively blocks DAG stimulation of channel exocytosis, and that DAG stimulates exocytosis of TRPC6-containing vesicles pre-docked to the plasma membrane. Mnuc13 family proteins play roles in the proper assembly of SNARE proteins and priming the vesicle competent for fusion. DAG stimulates TRPC6 exocytosis by targeting to the C1 domain of Munc13-2 (Xie et al. 2020).
References:
Abousaab, A., J. Warsi, M.S. Salker, and F. Lang. (2016). β-Klotho as a Negative Regulator of the Peptide Transporters PEPT1 and PEPT2. Cell Physiol Biochem 40: 874-882.
Akasaka-Manya, K., H. Manya, and T. Endo. (2016). Function and Change with Aging of α-Klotho in the Kidney. Vitam Horm 101: 239-256.
Almilaji, A., M. Sopjani, B. Elvira, J. Borras, M. Dërmaku-Sopjani, C. Munoz, J. Warsi, U.E. Lang, and F. Lang. (2014). Upregulation of the Creatine Transporter Slc6A8 by Klotho. Kidney Blood Press Res 39: 516-525.
Hu, M.C., M. Shi, and O.W. Moe. (2018). Role of αKlotho and FGF23 in regulation of type II Na-dependent phosphate co-transporters. Pflugers Arch. [Epub: Ahead of Print]
Ide, N., H. Olauson, T. Sato, M.J. Densmore, H. Wang, J.I. Hanai, T.E. Larsson, and B. Lanske. (2016). In vivo evidence for a limited role of proximal tubular Klotho in renal phosphate handling. Kidney Int 90: 348-362.
Kim, J.H., E.Y. Park, K.H. Hwang, K.S. Park, S.J. Choi, and S.K. Cha. (2021). Soluble αKlotho downregulates Orai1-mediated store-operated Ca entry via PI3K-dependent signaling. Pflugers Arch. [Epub: Ahead of Print]
Roig-Soriano, J., C. Sánchez-de-Diego, J. Esandi-Jauregui, S. Verdés, C.R. Abraham, A. Bosch, F. Ventura, and M. Chillón. (2023). Differential toxicity profile of secreted and processed α-Klotho expression over mineral metabolism and bone microstructure. Sci Rep 13: 4211.
Sinha, J., F. Chen, T. Miloh, R.C. Burns, Z. Yu, and B.L. Shneider. (2008). β-Klotho and FGF-15/19 inhibit the apical sodium-dependent bile acid transporter in enterocytes and cholangiocytes. Am. J. Physiol. Gastrointest Liver Physiol 295: G996-G1003.
Smith, E.R., S.G. Holt, and T.D. Hewitson. (2017). FGF23 activates injury-primed renal fibroblasts via FGFR4-dependent signalling and enhancement of TGF-β autoinduction. Int J Biochem. Cell Biol. 92: 63-78.
Sopjani, M. and M. Dërmaku-Sopjani. (2016). Klotho-Dependent Cellular Transport Regulation. Vitam Horm 101: 59-84.
Sopjani, M., M. Rinnerthaler, A. Almilaji, S. Ahmeti, and M. Dermaku-Sopjani. (2014). Regulation of cellular transport by klotho protein. Curr. Protein. Pept. Sci. 15: 828-835.
Warsi, J., A. Abousaab, and F. Lang. (2015). Up-Regulation of Excitatory Amino Acid Transporters EAAT1 and EAAT2 by ß-Klotho. Neurosignals 23: 59-70.
Xie, J., S.W. An, X. Jin, Y. Gui, and C.L. Huang. (2020). Munc13 mediates klotho-inhibitable diacylglycerol-stimulated exocytotic insertion of pre-docked TRPC6 vesicles. PLoS One 15: e0229799.
Xuan, N.T. and N.V. Hai. (2018). Changes in expression of klotho affect physiological processes, diseases, and cancer. Iran J Basic Med Sci 21: 3-8.
Zhu, L., H. Xie, Q. Liu, F. Ma, and H. Wu. (2021). Klotho inhibits H O -induced oxidative stress and apoptosis in periodontal ligament stem cells by regulating UCP2 expression. Clin Exp Pharmacol Physiol 48: 1412-1420.
Examples:
TC#	Name	Organismal Type	Example
8.A.49.1.1	The Klotho (KL; α-Klotho; alpha-Klotho) protein of 1012 aas and 2 (or more) TMSs, N- and C-terminal. Activates several transporters including the creatine transporter, CreaT or SLC6A8 (TC# 2.A.22.3.11; Almilaji et al. 2014) and the TRP6 cation channel (Smith et al. 2017). Klotho also regulates diverse calcium and potassium ion channels as well as several carriers including the Na⁺-coupled excitatory amino acid transporters EAAT3 and EAAT4, the Na⁺-coupled phosphate cotransporters, NaP_i-IIa and NaP_i-IIb, and a Na⁺/K⁺-ATPase. All these cellular transport regulations contribute in the aging suppressor role of Klotho (Sopjani et al. 2014). α-Klotho is associated with phosphate, vitamin D, and calcium homeostasis. The calcium imbalance in α-Klotho-negative mice may induce calpain overactivation, leading to cell death and tissue destruction. α-Klotho has glycosidase activity, capable of modifying the N-glycans of channels and transporters and regulating transmembrane movement of several ions, including calcium (Akasaka-Manya et al. 2016). It regulates diacylglycerol-stimulated exocytotic insertion of pre-docked TRPC6 vesicles (Xie et al. 2020). The aging-protective gene alpha-Klotho (KL) produces two main transcripts. The full-length mRNA generates a transmembrane protein that after proteolytic ectodomain shedding can be detected in serum as processed Klotho (p-KL), and a shorter transcript which codes for a putatively secreted protein (s-KL). Both isoforms exhibit potent pleiotropic beneficial properties (Roig-Soriano et al. 2023). s-KL (but not p-KL) is a safe therapeutic strategy to exploit KL anti-aging protective effects, presenting no apparent negative effects over mineral metabolism and bone microstructure (Roig-Soriano et al. 2023).	Animals	Klotho of Homo sapiens

8.A.49.1.2	β-Klotho of 1012 aas and 2 TMSs, N- and C-terminal. It is a β-glucuronidase which hydrolyzes 0-glycosyl bonds and binds fibroblast growth factor. Involved in several signalling pathways. Sometimes together with FGF, it regulates many channels and carriers including the Na⁺:bile acid co-transporter (TC# 2.A.28.1.5; Sinha et al. 2008) and the peptide transporters, PEPT1 and PEPT2 (Abousaab et al. 2016).	Animals	Beta-Klotho of Homo sapiens

8.A.49.1.3	Uncharacterized PspC domain-containing protein of 533 aas and 5 TMSs.		UP of Candidatus Saccharibacteria bacterium