Background/Aims: Klotho, a transmembrane protein expressed in chorioid plexus of the brain, kidney, and several other tissues, is required for inhibition of 1,25(OH)₂D₃ formation by FGF23. The extracellular domain of Klotho protein could be cleaved off, thus being released into blood or cerebrospinal fluid. At least in part by exerting β-glucuronidase activity, soluble klotho regulates several ion channels and carriers. Klotho protein deficiency accelerates the appearance of age related disorders including neurodegeneration and muscle wasting and eventually leads to premature death. The present study explored the effect of Klotho protein on the excitatory glutamate transporters EAAT1 (SLC1A3) and EAAT2 (SLC1A2), Na⁺ coupled carriers clearing excitatory amino acids from the synaptic cleft and thus participating in the regulation of neuronal excitability. Methods: cRNA encoding EAAT1 or EAAT2 was injected into Xenopus laevis oocytes and glutamate (2 mM)-induced inward current (I_Glu) taken as measure of glutamate transport. Measurements were made without or with prior 24 h treatment with soluble ß-Klotho protein (30 ng/ml) in the absence and presence of β-glucuronidase inhibitor D-saccharic acid 1,4-lactone monohydrate (DSAL,10 µM). Results: I_Glu was observed in EAAT1 and in EAAT2 expressing oocytes but not in water injected oocytes. In both, EAAT1 and EAAT2 expressing oocytes I_Glu was significantly increased by treatment with soluble ß-Klotho protein, an effect reversed by DSAL. Treatment with ß-klotho protein increased significantly the maximal transport rate without significantly modifying the affinity of the carriers. Conclusion: ß-Klotho up-regulates the excitatory glutamate transporters EAAT1 and EAAT2 and thus participates in the regulation of neuronal excitation.

Background/Aims: Activation of T cell receptors (TCRs) in CD4⁺ T cells leads to a cascade of signalling reactions including increase of intracellular calcium (Ca²⁺) levels with subsequent Ca²⁺ dependent stimulation of gene expression, proliferation, cell motility and cytokine release. The increase of cytosolic Ca²⁺ results from intracellular Ca²⁺ release with subsequent activation of store-operated Ca²⁺ entry (SOCE). Previous studies suggested miRNAs are required for the development and functions of CD4⁺ T cells. An enzyme called Dicer is required during the process of manufacturing mature miRNAs from the precursor miRNAs. In this study, we explored whether loss of Dicer in CD4⁺ T cells affects SOCE and thus Ca²⁺ dependent regulation of cellular functions. Methods: We tested the expression of Orai1 by q-RT-PCR and flow cytometry. Further, we measured SOCE by an inverted phase-contrast microscope with the Incident-light fluorescence illumination system using Fura-2. Intracellular Ca²⁺ was also measured by flow cytometry using Ca²⁺ sensitive dye Fluo-4. Results: We found that in Dicer deficient (Dicer^Δ/Δ) mice Orai1 was downregulated at mRNA and protein level in CD4⁺ T cells. Further, SOCE was significantly smaller in Dicer^Δ/Δ CD4⁺ T cells than in CD4⁺ T cells isolated from wild-type (Dicer^fl/fl) mice. Conclusion: Our data suggest that miRNAs are required for adequate Ca²⁺ entry into CD4⁺ T cells and thus triggering of Ca²⁺ sensitive immune functions.

Background/Aims: Janus kinase 3 (JAK3), a kinase mainly expressed in hematopoietic cells, has been shown to down-regulate the Na⁺/K⁺ ATPase and participate in the regulation of several ion channels and carriers. Channels expressed in thymus and regulating the abundance of T lymphocytes include the voltage gated K⁺ channel KCNE1/KCNQ1. The present study explored whether JAK3 contributes to the regulation of KCNE1/KCNQ1. Methods: cRNA encoding KCNE1/KCNQ1 was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild-type JAK3, constitutively active ^A568VJAK3, or inactive ^K851AJAK3. Voltage gated K⁺ channel activity was measured utilizing two electrode voltage clamp. Results: KCNE1/KCNQ1 activity was significantly increased by wild-type JAK3 and ^A568VJAK3, but not by ^K851AJAK3. The difference between oocytes expressing KCNE1/KCNQ1 alone and oocytes expressing KCNE1/KCNQ1 with ^A568VJAK3 was virtually abrogated by JAK3 inhibitor WHI-P154 (22 µM) but not by inhibition of transcription with actinomycin D (50 nM). Inhibition of KCNE1/KCNQ1 protein insertion into the cell membrane by brefeldin A (5 µM) resulted in a decline of the voltage gated current, which was similar in the absence and presence of ^A568VJAK3, suggesting that ^A568VJAK3 did not accelerate KCNE1/KCNQ1 protein retrieval from the cell membrane. Conclusion: JAK3 contributes to the regulation of membrane KCNE1/KCNQ1 activity, an effect sensitive to JAK3 inhibitor WHI-P154.

Background/Aims: β-Klotho, a transmembrane protein expressed in several tissues including the brain and the kidney, is critically important for inhibition of 1,25(OH)₂D₃ formation by FGF23. The extracellular domain of Klotho protein could be cleaved off, thus being released into blood or cerebrospinal fluid. Soluble klotho is a β-glucuronidase participating in the regulation of several ion channels and carriers. The present study explored the effect of β-Klotho protein on the peptide transporters PEPT1 and PEPT2. Methods: cRNA encoding PEPT1 or PEPT2 was injected into Xenopus laevis oocytes and glycine-glycine (2 mM)-induced inward current (I_Gly) taken as measure of glycine-glycine transport. Measurements were made without or with prior 24 h treatment with soluble β-Klotho protein (30 ng/ml) in the absence and presence of β-glucuronidase inhibitor D-saccharic acid 1,4-lactone monohydrate (DSAL,10 µM). Ussing chamber experiments were employed to determine electrogenic peptide transport across intestinal epithelia of klotho deficient (kl^-/-) and corresponding wild type (kl^+/+) mice. Results: I_Gly was observed in PEPT1 and in PEPT2 expressing oocytes but not in water injected oocytes. In both, PEPT1 and PEPT2 expressing oocytes I_Gly was significantly decreased by treatment with soluble β-Klotho protein. As shown for PEPT1, β-klotho protein decreased significantly the maximal transport rate without significantly modifying the affinity of the carrier. The effect of β-Klotho on PEPT1 was reversed by DSAL. Intestinal I_Gly was significantly larger in kl^-/- than in kl^+/+ mice. Conclusion: β-Klotho participates in the regulation of the peptide transporters PEPT1 and PEPT2.

Background: Cellular uptake of glutamate by the excitatory amino-acid transporters (EAATs) decreases excitation and thus participates in the regulation of neuroexcitability. Kinases impacting on neuronal function include Lithium-sensitive glycogen synthase kinase GSK3ß. The present study thus explored whether the activities of EAAT3 and/or EAAT4 isoforms are sensitive to GSK3ß. Methods: cRNA encoding wild type EAAT3 (SLC1A1) or EAAT4 (SLC1A6) was injected into Xenopus oocytes without or with additional injection of cRNA encoding wild type GSK3ß or the inactive mutant ^K85AGSK3ß. Dual electrode voltage clamp was performed in order to determine glutamate-induced current (I_EAAT). Results: Appreciable I_EAAT was observed in EAAT3 or EAAT4 expressing but not in water injected oocytes. I_EAAT was significantly increased by coexpression of GSK3ß but not by coexpression of ^K85AGSK3ß. Coexpression of GSK3ß increased significantly the maximal I_EAAT in EAAT3 or EAAT4 expressing oocytes, without significantly modifying apparent affinity of the carriers. Lithium (1 mM) exposure for 24 hours decreased I_EAAT in EAAT3 and GSK3ß expressing oocytes to values similar to I_EAAT in oocytes expressing EAAT3 alone. Lithium did not significantly modify I_EAAT in oocytes expressing EAAT3 without GSK3ß. Conclusions: Lithium-sensitive GSK3ß is a powerful regulator of excitatory amino acid transporters EAAT3 and EAAT4.