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      Regulation of the Voltage Gated K + Channel K v1.3 by Recombinant Human Klotho Protein

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          Abstract

          Background/Aims: Klotho, a protein mainly produced in the kidney and released into circulating blood, contributes to the negative regulation of 1,25(OH)<sub>2</sub>D<sub>3</sub> formation and is thus a powerful regulator of mineral metabolism. As β-glucuronidase, alpha Klotho protein further regulates the stability of several carriers and channels in the plasma membrane and thus regulates channel and transporter activity. Accordingly, alpha Klotho protein participates in the regulation of diverse functions seemingly unrelated to mineral metabolism including lymphocyte function. The present study explored the impact of alpha Klotho protein on the voltage gated K<sup>+</sup> channel K<sub>v1.3</sub>. Methods: cRNA encoding K<sub>v1.3</sub> (KCNA3) was injected into Xenopus oocytes and depolarization induced outward current in K<sub>v1.3</sub> expressing Xenopus oocytes determined utilizing dual electrode voltage clamp. Experiments were performed without or with prior treatment with recombinant human Klotho protein (50 ng/ml, 24 hours) in the absence or presence of a β-glucuronidase inhibitor D-saccharic acid-1,4-lactone (DSAL, 10 µM). Moreover, the voltage gated K<sup>+</sup> current was determined in Jcam lymphoma cells by whole cell patch clamp following 24 hours incubation without or with recombinant human Klotho protein (50 ng/ml, 24 hours). K<sub>v1.3</sub> protein abundance in Jcam cells was determined utilising fluorescent antibodies in flow cytometry. Results: In K<sub>v1.3</sub> expressing Xenopus oocytes the K<sub>v1.3</sub> currents and the protein abundance of K<sub>v1.3</sub> were both significantly enhanced after treatment with recombinant human Klotho protein (50 ng/ml, 24 hours), an effect reversed by presence of DSAL. Moreover, treatment with recombinant human Klotho protein increased K<sub>v</sub> currents and K<sub>v1.3</sub> protein abundance in Jcam cells. Conclusion: Alpha Klotho protein enhances K<sub>v1.3</sub> channel abundance and K<sub>v1.3</sub> currents in the plasma membrane, an effect depending on its β-glucuronidase activity.

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          Most cited references 73

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          Klotho: a novel phosphaturic substance acting as an autocrine enzyme in the renal proximal tubule.

          Klotho has profound effects on phosphate metabolism, but the mechanisms of how Klotho affects phosphate homeostasis is unknown. We detected Klotho in the proximal tubule cell, brush border, and urinary lumen, where phosphate homeostasis resides. Increasing Klotho in the kidney and urine chronically by transgenic overexpression or acutely by intravenous infusion caused hypophosphatemia, phosphaturia from decreased proximal phosphate reabsorption, and decreased activity and protein of the principal renal phosphate transporter NaPi-2a. The phosphaturic effect was present in FGF23-null mice, indicating a direct action distinct from Klotho's known role as a coreceptor for FGF23. Direct inhibition of NaPi-2a by Klotho was confirmed in cultured cells and in cell-free membrane vesicles characterized by acute inhibition of transport activity followed by decreased cell surface protein. Transport inhibition can be mimicked by recombinant beta-glucuronidase and is associated with proteolytic degradation and reduced surface NaPi-2a. The inhibitory effect of Klotho on NaPi-2a was blocked by beta-glucuronidase inhibitor but not by protease inhibitor. Klotho is a novel phosphaturic substance that acts as an enzyme in the proximal tubule urinary lumen by modifying glycans, which cause decreased transporter activity, followed by proteolytic degradation and possibly internalization of NaPi-2a from the apical membrane.
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            Secreted Klotho protein in sera and CSF: implication for post-translational cleavage in release of Klotho protein from cell membrane.

            Klotho mutant mice exhibit a set of phenotypes resembling human ageing. Although the function of Klotho remains unclear, mediation of its pleiotropic functions by putative humoral factor(s) has been presumed. Newly established antibodies against Klotho allowed the detection of secreted Klotho, a candidate for the putative humoral factor, in sera and cerebrospinal fluid. Surprisingly the secreted Klotho was 130 kDa, in contrast to the 70 kDa predicted form from klotho gene transcripts. The secreted as well as the membrane-bound Klotho proteins were suggested to form oligomerized complex. These results delineate post-translation processing of Klotho and possible regulatory mechanisms for secretion of Klotho in vivo.
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              The beta-glucuronidase klotho hydrolyzes and activates the TRPV5 channel.

              Blood calcium concentration is maintained within a narrow range despite large variations in dietary input and body demand. The Transient Receptor Potential ion channel TRPV5 has been implicated in this process. We report here that TRPV5 is stimulated by the mammalian hormone klotho. Klotho, a beta-glucuronidase, hydrolyzes extracellular sugar residues on TRPV5, entrapping the channel in the plasma membrane. This maintains durable calcium channel activity and membrane calcium permeability in kidney. Thus, klotho activates a cell surface channel by hydrolysis of its extracellular N-linked oligosaccharides.
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                Author and article information

                Journal
                KBR
                Kidney Blood Press Res
                10.1159/issn.1420-4096
                Kidney and Blood Pressure Research
                S. Karger AG
                1420-4096
                1423-0143
                2014
                December 2014
                15 December 2014
                : 39
                : 6
                : 609-622
                Affiliations
                aDepartment of Physiology, University of Tübingen, Tübingen, Germany; bInstitute of Physiology and Zurich Center for Integrative Human Physiology (ZIHP), University of Zurich, Zurich, Switzerland; cFaculty of Medicine, University of Prishtina, Prishtina, Kosova
                Author notes
                *Prof. Dr. med. Dr. h.c. Florian Lang, Physiologisches Institut der Universität Tübingen, Gmelinstr. 5, D-72076 Tübingen, (Germany), Tel. +49 7071 29 72194, Fax +49 7071 29 5618, E-Mail florian.lang@uni-tuebingen.de
                Article
                368472 Kidney Blood Press Res 2014;39:609-622
                10.1159/000368472
                25571875
                © 2015 S. Karger AG, Basel

                Open Access License: This is an Open Access article licensed under the terms of the Creative Commons Attribution-NonCommercial 3.0 Unported license (CC BY-NC) ( http://www.karger.com/OA-license), applicable to the online version of the article only. Distribution permitted for non-commercial purposes only. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

                Page count
                Pages: 14
                Categories
                Original Paper

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