Regulation of the Voltage Gated K⁺ Channel K_v1.3 by Recombinant Human Klotho Protein

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)₂D₃ 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⁺ channel K_v1.3. Methods: cRNA encoding K_v1.3 (KCNA3) was injected into Xenopus oocytes and depolarization induced outward current in K_v1.3 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⁺ 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_v1.3 protein abundance in Jcam cells was determined utilising fluorescent antibodies in flow cytometry. Results: In K_v1.3 expressing Xenopus oocytes the K_v1.3 currents and the protein abundance of K_v1.3 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_v currents and K_v1.3 protein abundance in Jcam cells. Conclusion: Alpha Klotho protein enhances K_v1.3 channel abundance and K_v1.3 currents in the plasma membrane, an effect depending on its β-glucuronidase activity.

Regulation of the Na⁺,Cl^- Coupled Creatine Transporter CreaT (SLC6A8) by the Janus Kinase JAK3

“…The data were filtered at 10 Hz and recorded with a Digidata 1322A A/D-D/A converter and Clampex.9.2 software for data acquisition (Axon Instruments). The analysis of the data was performed with Clampfit 9.2 (Axon Instruments) software [59,60]. The oocytes were maintained at 17°C in ND96-A solution containing: 88.5 mM NaCl, 2 mM KCl, 1 mM MgCl 2 , 1.8 mM CaCl 2 , 5 mM HEPES, 2.5 mM NaOH; Tretracycline (50 mg/l), Ciprofloxacin (1.6 mg/l), Refobacin (100 mg/l) and Theophylin (90 mg/l) as well as sodium pyruvate (5 mM) were added; pH was adjusted to 7.4 by addition of NaOH.…”

Up-Regulation of Excitatory Amino Acid Transporters EAAT1 and EAAT2 by ß-Klotho

“…10 ng cRNA encoding EAAT1 or EAAT2 were injected on the same day after preparation of the oocytes. The oocytes were maintained at 17°C in ND96-A, a solution containing (in mM): 88.5 NaCl, 2 KCl, 1 MgC1 2 , 1.8 CaC1 2 , 2.5 NaOH, 5 HEPES (pH 7.4), 5 sodium pyruvate (C 3 H 3 NaO 3 ), Gentamycin (100 mg/l), Tetracycline (50 mg/l), Ciprofloxacin (1.6 mg/l), and Theophiline (90 mg/l) [73,74]. Where indicated, 30 ng/ml ß-Klotho protein and/or 10 µM β-glucuronidase inhibitor DSAL were added to the respective solutions.…”

Up-Regulation of the Excitatory Amino Acid Transporters EAAT1 and EAAT2 by Mammalian Target of Rapamycin

“…Two-electrode voltage-clamp recordings were performed at a holding potential of -70 mV. The data were filtered at 10 Hz and recorded with a Digidata A/D-D/A converter (1322A Axon Instruments) and Clampex 9.2 software for data acquisition and analysis (Axon Instruments) [60][61][62]. The control superfusate contained 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2 and 5 mM HEPES, pH 7.4.…”