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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 Kv1.3. Methods: cRNA encoding Kv1.3 (KCNA3) was injected into Xenopus oocytes and depolarization induced outward current in Kv1.3 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). Kv1.3 protein abundance in Jcam cells was determined utilising fluorescent antibodies in flow cytometry. Results: In Kv1.3 expressing Xenopus oocytes the Kv1.3 currents and the protein abundance of Kv1.3 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 Kv currents and Kv1.3 protein abundance in Jcam cells. Conclusion: Alpha Klotho protein enhances Kv1.3 channel abundance and Kv1.3 currents in the plasma membrane, an effect depending on its β-glucuronidase activity.

Background/Aims: Mineralocorticoid excess leads to vascular injury, which is partially due to hypertension but in addition involves increased concentration of cytosolic Ca2+ concentration in platelets, key players in the pathophysiology of occlusive vascular disease. Mineralocorticoids are in part effective by rapid nongenomic mechanisms including phosphatidylinositide-3-kinase (PI3K) signaling, which involves activation of the serum & glucocorticoid inducible kinase (SGK) isoforms. SGK1 has in turn been shown to participate in the regulation of the pore forming Ca2+ channel protein Orai1 in platelets. Orai1 accomplishes entry of Ca2+, which is in turn known to trigger cell membrane scrambling. Platelets lack nuclei but are able to express protein by translation, which is stimulated by PI3K signaling. The present study explored whether the mineralocorticoid desoxycorticosterone acetate (DOCA) influences platelet Orai1 protein abundance, cytosolic Ca2+-activity ([Ca2+]i), phosphatidylserine abundance at the cell surface and/or cell volume. Methods: Orai1 protein abundance was estimated utilizing CF™488A conjugated antibodies, [Ca2+]i utilizing Fluo3-fluorescence, phosphatidylserine abundance utilizing FITC-labelled annexin V, and cell volume utilizing forward scatter in flow cytometry. Results: DOCA (10 µg/ml) treatment of murine platelets was followed by a significant increase of Orai1 protein abundance, [Ca2+]i, percentage of phosphatidylserine exposing platelets and platelet swelling. The effect on [Ca2+]i, phosphatidylserine abundance and cell volume were completely abrogated by addition of the specific SGK inhibitor EMD638683 (50 µM) Conclusions: The mineralocorticoid DOCA upregulates Orai1 protein abundance in the cell membrane, thus increasing [Ca2+]i and triggering phosphatidylserine abundance, effects paralleled by platelet swelling.