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Type 1 diabetes (T1D) results from autoimmune destruction of β-cells in the pancreas. Protein tyrosine phosphatases (PTPs) are candidate genes for T1D and play a key role in autoimmune disease development and β-cell dysfunction. Here, we assessed the global protein and individual PTP profiles in the pancreas from nonobese mice with early-onset diabetes (NOD) mice treated with an anti-CD3 monoclonal antibody and interleukin-1 receptor antagonist. The treatment reversed hyperglycemia, and we observed enhanced expression of PTPN2, a PTP family member and T1D candidate gene, and endoplasmic reticulum (ER) chaperones in the pancreatic islets. To address the functional role of PTPN2 in β-cells, we generated PTPN2-deficient human stem cell-derived β-like and EndoC-βH1 cells. Mechanistically, we demonstrated that PTPN2 inactivation in β-cells exacerbates type I and type II interferon signaling networks and the potential progression toward autoimmunity. Moreover, we established the capacity of PTPN2 to positively modulate the Ca2+-dependent unfolded protein response and ER stress outcome in β-cells. Adenovirus-induced overexpression of PTPN2 partially protected from ER stress-induced β-cell death. Our results postulate PTPN2 as a key protective factor in β-cells during inflammation and ER stress in autoimmune diabetes. [ABSTRACT FROM AUTHOR]

Most obese and insulin-resistant individuals do not develop diabetes. This is the result of the capacity of β-cells to adapt and produce enough insulin to cover the needs of the organism. The underlying mechanism of β-cell adaptation in obesity, however, remains unclear. Previous studies have suggested a role for STAT3 in mediating β-cell development and human glucose homeostasis, but little is known about STAT3 in β-cells in obesity. We observed enhanced cytoplasmic expression of STAT3 in severely obese subjects with diabetes. To address the functional role of STAT3 in adult β-cells, we generated mice with tamoxifen-inducible partial or full deletion of STAT3 in β-cells and fed them a high-fat diet before analysis. Interestingly, β-cell heterozygous and homozygous STAT3-deficient mice showed glucose intolerance when fed a high-fat diet. Gene expression analysis with RNA sequencing showed that reduced expression of mitochondrial genes in STAT3 knocked down human EndoC-β1H cells, confirmed in FACS-purified β-cells from obese STAT3-deficient mice. Moreover, silencing of STAT3 impaired mitochondria activity in EndoC-β1H cells and human islets, suggesting a mechanism for STAT3-modulated β-cell function. Our study postulates STAT3 as a novel regulator of β-cell function in obesity. [ABSTRACT FROM AUTHOR]

Background: MicroRNAs (miRNAs) negatively regulate gene expression at a posttranscriptional level. Dicer, a cytoplasmic RNase III enzyme, is required for the maturation of miRNAs from precursor miRNAs. Dicer, therefore, is a critical enzyme involved in the biogenesis and processing of miRNAs. Several biological processes are controlled by miRNAs, including the regulation of T cell development and function. T cells generate reactive oxygen species (ROS) with parallel H+ extrusion accomplished by the Na+/H+-exchanger 1 (NHE1). The present study explored whether ROS production, as well as NHE1 expression and function are sensitive to the lack of Dicer (miRNAs deficient) and could be modified by individual miRNAs. Methods: CD4+ T cells were isolated from CD4 specific Dicer deficient (DicerΔ/Δ) mice and the respective control mice (Dicerfl/fl). Transcript and protein levels were quantified with RT-PCR and Western blotting, respectively. For determination of intracellular pH (pHi) cells were incubated with the pH sensitive dye bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) and Na+/ H+ exchanger (NHE) activity was calculated from re-alkalinization after an ammonium pulse. Changes in cell volume were measured using the forward scatter in flow cytometry, and ROS production utilizing 2',7' -dichlorofluorescin diacetate (DCFDA) fluorescence. Transfection of miRNA-control and mimics in T cells was performed using DharmaFECT3 reagent. Results: ROS production, cytosolic H+ concentration, NHE1 transcript and protein levels, NHE activity, and cell volume were all significantly higher in CD4+ T cells from DicerΔ/Δ mice than in CD4+ T cells from Dicerfl/fl mice. Furthermore, individual miR-200b and miR-15b modify pHi and NHE activity in Dicerfl/fl and DicerΔ/Δ CD4+ T cells, respectively. Conclusions: Lack of Dicer leads to oxidative stress, cytosolic acidification, upregulated NHE1 expression and activity as well as swelling of CD4+ T cells, functions all reversed by miR-15b or miR-200b [ABSTRACT FROM AUTHOR]

Excitatory amino acid transporters EAAT1 (SLC1A3), EAAT2 (SLC1A2), EAAT3 (SLC1A1), and EAAT4 (SLC1A6) serve to clear L-glutamate from the synaptic cleft and are thus important for the limitation of neuronal excitation. EAAT3 has previously been shown to form complexes with caveolin-1, a major component of caveolae, which participate in the regulation of transport proteins. The present study explored the impact of caveolin-1 on electrogenic transport by excitatory amino acid transporter isoforms EAAT1-4. To this end cRNA encoding EAAT1, EAAT2, EAAT3, or EAAT4 was injected into Xenopus oocytes without or with additional injection of cRNA encoding caveolin-1. The L-glutamate (2 mM)-induced inward current (I Glu) was taken as a measure of glutamate transport. As a result, I Glu was observed in EAAT1-, EAAT2-, EAAT3-, or EAAT4-expressing oocytes but not in water-injected oocytes, and was significantly decreased by coexpression of caveolin-1. Caveolin-1 decreased significantly the maximal transport rate. Treatment of EAATs-expressing oocytes with brefeldin A (5 µM) was followed by a decrease in conductance, which was similar in oocytes expressing EAAT together with caveolin-1 as in oocytes expressing EAAT1-4 alone. Thus, caveolin-1 apparently does not accelerate transporter protein retrieval from the cell membrane. In conclusion, caveolin-1 is a powerful negative regulator of the excitatory glutamate transporters EAAT1, EAAT2, EAAT3, and EAAT4. [ABSTRACT FROM AUTHOR]

Background/Aims: The oxidative stress-responsive kinase 1 (OSR1) and the serine/threonine kinases SPAK (SPS1-related proline/alanine-rich kinase) are under the control of WNK (withno- K [Lys]) kinases. OSR1 and SPAK participate in diverse functions including cell volume regulation and neuronal excitability. Cell volume and neuronal excitation are further modified by the large conductance Ca2+-activated K+ channels (maxi K+ channel or BK channels). An influence of OSR1 and/or SPAK on BK channel activity has, however, never been shown. The present study thus explored whether OSR1 and/or SPAK modify the activity of BK channels. Methods: cRNA encoding the Ca2+ insensitive BK channel mutant BKM513I+Δ899-903 was injected into Xenopus laevis oocytes without or with additional injection of cRNA encoding wild-type OSR1 or wild-type SPAK, constitutively activeT185EOSR1, catalytically inactive D164AOSR1, constitutively active T233ESPAK or catalytically inactive D212ASPAK. K+ channel activity was measured utilizing dual electrode voltage clamp. Results: BK channel activity in BKM513I+Δ899-903 expressing oocytes was significantly decreased by co-expression of OSR1 or SPAK. The effect of wild-type OSR1/SPAK was mimicked by T185EOSR1 and T233ESPAK, but not by D164AOSR1 or D212ASPAK. Conclusions: OSR1 and SPAK suppress BK channels, an effect possibly contributing to cell volume regulation and neuroexcitability. [ABSTRACT FROM AUTHOR]

The tyrosine kinase Janus kinase 3 (JAK3) participates in the regulation of cell proliferation and apoptosis. The kinase further influences ion channels and transport proteins. The present study explored whether JAK3 contributes to the regulation of the voltage-gated K(+) channel Kv1.5, which participates in the regulation of diverse functions including atrial cardiac action potential and tumor cell proliferation. To this end, cRNA encoding Kv1.5 was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild-type JAK3, constitutively active (A568V)JAK3, or inactive (K851A)JAK3. Voltage-gated K(+) channel activity was measured utilizing dual electrode voltage clamp, and Kv1.5 channel protein abundance in the cell membrane was quantified utilizing chemiluminescence of Kv1.5 containing an extracellular hemagglutinin epitope (Kv1.5-HA). As a result, Kv1.5 activity and Kv1.5-HA protein abundance were significantly decreased by wild-type JAK3 and (A568V)JAK3, but not by (K851A)JAK3. Inhibition of Kv1.5 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 (A568V)JAK3, suggesting that (A568V)JAK3 did not accelerate Kv1.5 protein retrieval from the cell membrane. A 24 h treatment with ouabain (100 µM) significantly decreased the voltage-gated current in oocytes expressing Kv1.5 without or with (A568V)JAK3 and dissipated the difference between oocytes expressing Kv1.5 alone and oocytes expressing Kv1.5 with (A568V)JAK3. In conclusion, JAK3 contributes to the regulation of membrane Kv1.5 protein abundance and activity, an effect sensitive to ouabain and thus possibly involving Na(+)/K(+) ATPase activity. [ABSTRACT FROM AUTHOR]

SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1) are cell volume-sensitive kinases regulated by WNK (with-no-K[Lys]) kinases. SPAK/OSR1 regulate several channels and carriers. SPAK/OSR1 sensitive functions include neuronal excitability. Orchestration of neuronal excitation involves the excitatory glutamate transporters EAAT1 and EAAT2. Sensitivity of those carriers to SPAK/OSR1 has never been shown. The present study thus explored whether SPAK and/or OSR1 contribute to the regulation of EAAT1 and/or EAAT2. To this end, cRNA encoding EAAT1 or EAAT2 was injected into Xenopus oocytes without or with additional injection of cRNA encoding wild-type SPAK or wild-type OSR1, constitutively active (T233E)SPAK, WNK insensitive (T233A)SPAK, catalytically inactive (D212A)SPAK, constitutively active (T185E)OSR1, WNK insensitive (T185A)OSR1 or catalytically inactive (D164A)OSR1. The glutamate (2 mM)-induced inward current (I Glu) was taken as a measure of glutamate transport. As a result, I Glu was observed in EAAT1- and in EAAT2-expressing oocytes but not in water-injected oocytes, and was significantly decreased by coexpression of SPAK and OSR1. As shown for EAAT2, SPAK, and OSR1 decreased significantly the maximal transport rate but significantly enhanced the affinity of the carrier. The effect of wild-type SPAK/OSR1 on EAAT1 and EAAT2 was mimicked by (T233E)SPAK and (T185E)OSR1, but not by (T233A)SPAK, (D212A)SPAK, (T185A)OSR1, or (D164A)OSR1. Coexpression of either SPAK or OSR1 decreased the EAAT2 protein abundance in the cell membrane of EAAT2-expressing oocytes. In conclusion, SPAK and OSR1 are powerful negative regulators of the excitatory glutamate transporters EAAT1 and EAAT2. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: The voltage gated K+ channels Kv1.3 and Kv1.5 contribute to the orchestration of cell proliferation. Kinases participating in the regulation of cell proliferation include protein kinase B (PKB/Akt). The present study thus explored whether PKB/Akt modifies the abundance and function of Kv1.3 and Kv1.5. Methods: Kv1.3 or Kv1.5 was expressed in Xenopus laevis oocytes with or without wild-type PKB/Akt, constitutively active T308D/S473DPKB/Akt or inactive T308A/S473APKB/Akt. The channel activity was quantified utilizing dual electrode voltage clamp. Moreover, HAtagged Kv1.5 protein was determined utilizing chemiluminescence. Results: Voltage gated K+ currents were observed in Kv1.3 or Kv1.5 expressing oocytes but not in water-injected oocytes or in oocytes expressing PKB/Akt alone. Co-expression of PKB/Akt or T308D/S473DPKB/Akt, but not coexpression of T308A/S473APKB/Akt significantly increased the voltage gated current in both Kv1.3 and Kv1.5 expressing oocytes. As shown for Kv1.5, co-expression of PKB/Akt enhanced the channel protein abundance in the cell membrane. In Kv1.5 expressing oocytes voltage gated current decreased following inhibition of carrier insertion by brefeldin A (5 µM) to similarly low values in the absence and presence of PKB/Akt, suggesting that PKB/Akt stimulated carrier insertion into rather than inhibiting carrier retrieval from the cell membrane. Conclusion: PKB/Akt up-regulates both, Kv1.3 and Kv1.5 K+ channels. [ABSTRACT FROM AUTHOR]

Background/Aims: SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1), kinases controlled by WNK (with-no-K[Lys] kinase), are powerful regulators of cellular ion transport and blood pressure. Observations in gene-targeted mice disclosed an impact of SPAK/OSR1 on phosphate metabolism. The present study thus tested whether SPAK and/or OSR1 contributes to the regulation of the intestinal Na+-coupled phosphate co-transporter NaPi-IIb (SLC34A2). Methods: cRNA encoding NaPi-IIb was injected into Xenopus laevis oocytes without or with additional injection of cRNA encoding wild-type SPAK, constitutively active T233ESPAK, WNK insensitive T233ASPAK, catalytically inactive D212ASPAK, wild-type OSR1, constitutively active T185EOSR1, WNK insensitive T185AOSR1 or catalytically inactive D164AOSR1. The phosphate (1 mM)-induced inward current (IPi) was taken as measure of phosphate transport. Results: IPi was observed in NaPi-IIb expressing oocytes but not in water injected oocytes, and was significantly increased by co-expression of SPAK, T233ESPAK, OSR1, T185EOSR1 or SPAK+OSR1, but not by co-expression of T233ASPAK, D212ASPAK, T185AOSR1, or D164AOSR1. SPAK and OSR1 both increased the maximal transport rate of the carrier. Conclusions: SPAK and OSR1 are powerful stimulators of the intestinal Na+-coupled phosphate co-transporter NaPi-IIb. [ABSTRACT FROM AUTHOR]

Background/Aims: Kir2.1 (KCNJ2) channels are expressed in neurons, skeletal muscle and cardiac tissue and maintain the resting membrane potential. The activity of those channels is regulated by diverse signalling molecules. The present study explored whether Kir2.1 channels are sensitive to the transporter and channels regulating kinases SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1), which are in turn regulated by WNK (with-no-K[Lys]) kinases. Methods: cRNA encoding Kir2.1 was injected into Xenopus laevis oocytes with or without additional injection of cRNA encoding wild-type SPAK, constitutively active T233ESPAK, WNK insensitive T233ASPAK, catalytically inactive D212ASPAK, wild-type OSR1, constitutively active T185EOSR1, WNK insensitive T185AOSR1 and catalytically inactive D164AOSR1. Inwardly rectifying K+ channel activity was quantified utilizing dual electrode voltage clamp and Kir2.1 channel protein abundance in the cell membrane was measured utilizing chemiluminescence of Kir2.1 containing an extracellular HA-tag epitope. Results: Kir2.1 activity was significantly enhanced by wild-type SPAK and T233ESPAK, but not by T233ASPAK and D212ASPAK, as well as by wild-type OSR1 and T185EOSR1, but not by T185AOSR1 and D164AOSR1. As shown for SPAK, the kinases enhanced Kir2.1 protein abundance in the cell membrane. The difference of current and conductance between oocytes expressing Kir2.1 together with SPAK or OSR1 and oocytes expressing Kir2.1 alone was dissipated following a 24 hours inhibition of channel insertion into the cell membrane by brefeldin A (5 µM). Conclusions: SPAK and OSR1 are both stimulators of Kir2.1 activity. They are presumably effective by enhancing channel insertion into the cell membrane. © 2015 The Author(s) Published by S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background/Aims: KCNQ1/E1 channels are expressed in diverse tissues and serve a variety of functions including endolymph secretion in the inner ear, cardiac repolarization, epithelial transport and cell volume regulation. Kinases involved in regulation of epithelial transport and cell volume include SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1), which are under control of WNK (with-no-K[Lys]) kinases. The present study explored whether KCNQ1/E1 channels are regulated by SPAK and/or OSR1. Methods: cRNA encoding KCNQ1/E1 was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild-type SPAK, constitutively active T233ESPAK, WNK insensitive T233ASPAK, catalytically inactive D212ASPAK, wild-type OSR1, constitutively active T185EOSR1, WNK insensitive T185AOSR1 and catalytically inactive D164AOSR1. Voltage gated K+ channel activity was quantified utilizing dual electrode voltage clamp and KCNQ1/E1 channel protein abundance in the cell membrane utilizing chemiluminescence of KCNQ1/E1 containing an extracellular Flag tag epitope (KCNQ1-Flag/E1). Results: KCNQ1/E1 activity and KCNQ1-Flag/E1 protein abundance were significantly enhanced by wild-type SPAK and T233ESPAK, but not by T233ASPAK and D212ASPAK. Similarly, KCNQ1/E1 activity and KCNQ1-Flag/E1 protein abundance were significantly increased by wild-type OSR1 and T185EOSR1, but not by T185AOSR1 and D164AOSR1. Conclusions: SPAK and OSR1 participate in the regulation of KCNQ1/E1 protein abundance and activity. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Checkpoint kinase 2 (Chk2) is the main effector kinase of ataxia telangiectasia mutated (ATM) and responsible for cell cycle regulation. ATM signaling has been shown to upregulate interferon-regulating factor-1 (IRF-1), a transcription factor also expressed in the kidney. Calcitriol (1,25 (OH)D), a major regulator of mineral metabolism, is generated by 25-hydroxyvitamin D 1α-hydroxylase in the kidney. Since 25-hydroxyvitamin D 1α-hydroxylase expression is enhanced by IRF-1, the present study explored the role of Chk2 for calcitriol formation and mineral metabolism. Chk2-deficient mice ( chk2) were compared to wild-type mice ( chk2). Transcript levels of renal 25-hydroxyvitamin D 1α-hydroxylase, Chk2, and IRF-1 were determined by RT-PCR; Klotho expression by Western blotting; bone density by μCT analysis; serum or plasma 1,25 (OH)D, PTH, and C-terminal FGF23 concentrations by immunoassays; and serum, fecal, and urinary calcium and phosphate concentrations by photometry. The renal expression of IRF-1 and 25-hydroxyvitamin D 1α-hydroxylase as well as serum 1,25 (OH)D and FGF23 levels were significantly lower in chk2 mice compared to chk2 mice. Plasma PTH was not different between the genotypes. Renal calcium and phosphate excretion were significantly higher in chk2 mice than in chk2 mice despite hypophosphatemia and normocalcemia. Bone density was not different between the genotypes. We conclude that Chk2 regulates renal 25-hydroxyvitamin D 1α-hydroxylase expression thereby impacting on calcium and phosphate metabolism. [ABSTRACT FROM AUTHOR]

Background/Aims: Janus kinase 3 (JAK3), a tyrosine kinase contributing to the regulation of cell proliferation and apoptosis of lymphocytes and tumour cells, has been shown to modify the expression and function of several ion channels and transport proteins. Channels involved in the regulation of cell proliferation include the large conductance voltage- and Ca2+-activated K+ channel BK. The present study explored whether JAK3 modifies BK channel protein abundance and current. Methods: cRNA encoding Ca2+-insensitive BK channel (BKM513I+Δ899-903) 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 dual electrode voltage clamp. Moreover, BK channel protein abundance was determined utilizing flow cytometry in CD19+ B lymphocyte cell membranes from mice lacking functional JAK3 (jak3-/-) and corresponding wild-type mice (jak3+/+). Results: BK activity in BKM513I+Δ899-903 expressing oocytes was slightly but significantly decreased by coexpression of wild-type JAK3 and of A568VJAK3, but not by coexpression of K851AJAK3. The BK channel protein abundance in the cell membrane was significantly higher in jak3-/- than in jak3+/+ B lymphocytes. The decline of conductance in BK and JAK3 coexpressing oocytes following inhibition of channel protein insertion by brefeldin A (5 µM) was similar in oocytes expressing BK with JAK3 and oocytes expressing BK alone, indicating that JAK3 might slow channel protein insertion into rather than accelerating channel protein retrieval from the cell membrane. Conclusion: JAK3 is a weak negative regulator of membrane BK protein abundance and activity. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background/Aims: The WNK-dependent STE20/SPS1-related proline/alanine-rich kinase SPAK participates in the regulation of NaCl and Na+,K+,2Cl- cotransport and thus renal salt excretion. The present study explored whether SPAK has similarly the potential to regulate the epithelial Na+ channel (ENaC). Methods: ENaC was expressed in Xenopus oocytes with or without additional expression of wild type SPAK, constitutively active T233ESPAK, WNK insensitive T233ASPAK or catalytically inactive D212ASPAK, and ENaC activity estimated from amiloride (50 µM) sensitive current (Iamil) in dual electrode voltage clamp experiments. Moreover, Ussing chamber was employed to determine Iamil in colonic tissue from wild type mice (spakwt/wt) and from gene targeted mice carrying WNK insensitive SPAK (spaktg/tg Results: Iamil was observed in ENaC-expressing oocytes, but not in water-injected oocytes. In ENaC expressing oocytes Iamil was significantly increased following coexpression of wild-type SPAK and T233ESPAK, but not following coexpression of T233ASPAK or D212ASPAK. Colonic Iamil was significantly higher in spakw wtthan in spaktg/tg mice. Conclusion: SPAK has the potential to up-regulate ENaC. [ABSTRACT FROM AUTHOR]

USP18 (Ubiquitin-like specific protease 18) is an enzyme cleaving ubiquitin from target proteins. USP18 plays a pivotal role in antiviral and antibacterial immune responses. On the other hand, ubiquitination participates in the regulation of several ion channels and transporters. USP18 sensitivity of transporters has, however, never been reported. The present study thus explored, whether USP18 modifies the activity of the peptide transporters PEPT1 and PEPT2, and whether the peptide transporters are sensitive to the ubiquitin ligase Nedd4-2. To this end, cRNA encoding PEPT1 or PEPT2 was injected into Xenopus laevis oocytes without or with additional injection of cRNA encoding USP18. Electrogenic peptide (glycine-glycine) transport was determined by dual electrode voltage clamp. As a result, in Xenopus laevis oocytes injected with cRNA encoding PEPT1 or PEPT2, but not in oocytes injected with water or with USP18 alone, application of the dipeptide gly-gly (2 mM) was followed by the appearance of an inward current (Igly-gly). Coexpression of USP18 significantly increased Igly-gly in both PEPT1 and PEPT2 expressing oocytes. Kinetic analysis revealed that coexpression of USP18 increased maximal Igly-gly. Conversely, overexpression of the ubiquitin ligase Nedd4-2 decreased Igly-gly. Coexpression of USP30 similarly increased Igly-gly in PEPT1 expressing oocytes. In conclusion, USP18 sensitive cellular functions include activity of the peptide transporters PEPT1 and PEPT2. [ABSTRACT FROM AUTHOR]

Gene variants of the leucine-rich repeat kinase 2 (LRRK2) are associated with susceptibility to Parkinson's disease (PD). Besides brain and periphery, LRRK2 is expressed in various immune cells including dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity. However, the function of LRRK2 in the immune system is still incompletely understood. Here, Ca2+-signaling was analyzed in DCs isolated from gene-targeted mice lacking lrrk2 (Lrrk2-/-) and their wild-type littermates (Lrrk2+/+). According to Western blotting, Lrrk2 was expressed in Lrrk2+/+ DCs but not in Lrrk2-/-DCs. Cytosolic Ca2+ levels ([Ca2+]i) were determined utilizing Fura-2 fluorescence and whole cell currents to decipher electrogenic transport. The increase of [Ca2+]i following inhibition of sarcoendoplasmatic Ca2+-ATPase with thapsigargin (1 mM) in the absence of extracellular Ca2+ (Ca2+-release) and the increase of [Ca2+]i following subsequent readdition of extracellular Ca2+ (SOCE) were both significantly larger in Lrrk2-/- than in Lrrk2+/+ DCs. The augmented increase of [Ca2+]i could have been due to impaired Ca2+ extrusion by K+-independent (NCX) and/or K+-dependent (NCKX) Na+/Ca2+-exchanger activity, which was thus determined from the increase of [Ca2+]i, (D[Ca2+]i), and current following abrupt replacement of Na+ containing (130 mM) and Ca2+ free (0 mM) extracellular perfusate by Na+ free (0mM) and Ca2+ containing (2 mM) extracellular perfusate. As a result, both slope and peak of D[Ca2+]i as well as Na+/Ca2+ exchanger-induced current were significantly lower in Lrrk2-/- than in Lrrk2+/+ DCs. A 6 or 24 hour treatment with the LRRK2 inhibitor GSK2578215A (1 µM) significantly decreased NCX1 and NCKX1 transcript levels, significantly blunted Na+/Ca2+- exchanger activity, and significantly augmented the increase of [Ca2+]i following Ca2+-release and SOCE. In conclusion, the present observations disclose a completely novel functional significance of LRRK2, i.e., the up-regulation of Na+/Ca2+ exchanger transcription and activity leading to attenuation of Ca2+-signals in DCs. [ABSTRACT FROM AUTHOR]

Parvovirus B19 (B19V) has previously been shown to cause endothelial dysfunction. B19V capsid protein VP1 harbors a lysophosphatidylcholine producing phospholipase A2 (PLA2). Lysophosphatidylcholine inhibits Na(+)/K(+) ATPase, which in turn may impact on the activity of inwardly rectifying K(+) channels. The present study explored whether VP1 modifies the activity of Kir2.1 K(+) channels. cRNA encoding Kir2.1 was injected into Xenopus oocytes without or with cRNA encoding VP1 isolated from a patient suffering from fatal B19V-induced inflammatory cardiomyopathy or the VP1 mutant (H153A)VP1 lacking a functional PLA2 activity. K(+) channel activity was determined by dual electrode voltage clamp. In addition, Na(+)/K(+)-ATPase activity was estimated from K(+)-induced pump current (I(pump)) and ouabain-inhibited current (I(ouabain)). Injection of cRNA encoding Kir2.1 into Xenopus oocytes was followed by appearance of inwardly rectifying K(+) channel activity (I(K)), which was significantly decreased by additional injection of cRNA encoding VP1, but not by additional injection of cRNA encoding (H153A)VP1. The effect of VP1 on I K was mimicked by lysophosphatidylcholine (1 μg/ml) and by inhibition of Na(+)/K(+)-ATPase with 0.1 mM ouabain. In the presence of lysophosphatidylcholine, I K was not further decreased by additional treatment with ouabain. The B19V capsid protein VP1 thus inhibits Kir2.1 channels, an effect at least partially due to PLA2-dependent formation of lysophosphatidylcholine with subsequent inhibition of Na(+)/K(+)-ATPase activity. [ABSTRACT FROM AUTHOR]

Parvovirus B19 (B19V) has previously been shown to cause endothelial dysfunction. B19V capsid protein VP1 harbors a lysophosphatidylcholine producing phospholipase A2 (PLA2). Lysophosphatidylcholine inhibits Na/K ATPase, which in turn may impact on the activity of inwardly rectifying K channels. The present study explored whether VP1 modifies the activity of Kir2.1 K channels. cRNA encoding Kir2.1 was injected into Xenopus oocytes without or with cRNA encoding VP1 isolated from a patient suffering from fatal B19V-induced inflammatory cardiomyopathy or the VP1 mutant VP1 lacking a functional PLA2 activity. K channel activity was determined by dual electrode voltage clamp. In addition, Na/K-ATPase activity was estimated from K-induced pump current ( I) and ouabain-inhibited current ( I). Injection of cRNA encoding Kir2.1 into Xenopus oocytes was followed by appearance of inwardly rectifying K channel activity ( I), which was significantly decreased by additional injection of cRNA encoding VP1, but not by additional injection of cRNA encoding VP1. The effect of VP1 on I was mimicked by lysophosphatidylcholine (1 μg/ml) and by inhibition of Na/K-ATPase with 0.1 mM ouabain. In the presence of lysophosphatidylcholine, I was not further decreased by additional treatment with ouabain. The B19V capsid protein VP1 thus inhibits Kir2.1 channels, an effect at least partially due to PLA2-dependent formation of lysophosphatidylcholine with subsequent inhibition of Na/K-ATPase activity. [ABSTRACT FROM AUTHOR]

The janus-activated kinase 2 JAK2 participates in the signalling of several hormones including interferon, a powerful regulator of lymphocyte function. Lymphocyte activity and survival depend on the activity of the voltage-gated K channel KCNA3 (Kv1.3). The present study thus explored whether JAK2 modifies the activity of voltage-gated K channel KCNA3. To this end, cRNA encoding KCNA3 was injected in Xenopus oocytes with or without additional injection of cRNA encoding wild-type human JAK2, human inactive JAK2 mutant, or human gain-of-function JAK2 mutant. KCNA3-dependent depolarization-induced current was determined utilizing dual-electrode voltage clamp, and protein KCNA3 abundance in the cell membrane was quantified by chemiluminescence. Moreover, the effect of interferon-γ on voltage-gated K current was determined by patch clamp in mainly KCNA3-expressing Jurkat T cells with or without prior treatment with JAK2 inhibitor AG490 (40 µM). As a result, KCNA3 channel activity and protein abundance were up-regulated by coexpression of JAK2 or JAK2 but not JAK2. The effect of JAK2 coexpression was reversed by AG490 treatment. In human Jurkat T lymphoma cells, voltage-gated K current was up-regulated by interferon-γ and down-regulated by AG490 (40 µM). In conclusion, JAK2 participates in the signalling, regulating the voltage-gated K channel KCNA3. [ABSTRACT FROM AUTHOR]

SPS1-related proline/alanine-rich kinase (SPAK) and oxidative stress-responsive kinase 1 (OSR1) are potent regulators of several transporters and ion channels. The kinases are under regulation of with-no-K(Lys) (WNK) kinases. The present study explored whether SPAK and/or OSR1 modify the expression and/or activity of the voltage-gated K(+) channel Kv1.5, which participates in the regulation of diverse functions including atrial cardiac action potential and tumor cell proliferation. cRNA encoding Kv1.5 was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild-type SPAK, constitutively active (T233E)SPAK, WNK insensitive (T233A)SPAK, catalytically inactive (D212A)SPAK, wild-type OSR1, constitutively active (T185E)OSR1, WNK insensitive (T185A)OSR1, and catalytically inactive (D164A)OSR1. Voltage-gated K(+) channel activity was quantified utilizing dual electrode voltage clamp and Kv1.5 channel protein abundance in the cell membrane utilizing chemiluminescence of Kv1.5 containing an extracellular hemagglutinin epitope (Kv1.5-HA). Kv1.5 activity and Kv1.5-HA protein abundance were significantly decreased by wild-type SPAK and (T233E)SPAK, but not by (T233A)SPAK and (D212A)SPAK. Similarly, Kv1.5 activity and Kv1.5-HA protein abundance were significantly down-regulated by wild-type OSR1 and (T185E)OSR1, but not by (T185A)OSR1 and (D164A)OSR1. Both, SPAK and OSR1 decrease cell membrane Kv1.5 protein abundance and activity. [ABSTRACT FROM AUTHOR]

SPS1-related proline/alanine-rich kinase (SPAK) and oxidative stress-responsive kinase 1 (OSR1) are potent regulators of several transporters and ion channels. The kinases are under regulation of with-no-K(Lys) (WNK) kinases. The present study explored whether SPAK and/or OSR1 modify the expression and/or activity of the voltage-gated K channel Kv1.5, which participates in the regulation of diverse functions including atrial cardiac action potential and tumor cell proliferation. cRNA encoding Kv1.5 was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild-type SPAK, constitutively active SPAK, WNK insensitive SPAK, catalytically inactive SPAK, wild-type OSR1, constitutively active OSR1, WNK insensitive OSR1, and catalytically inactive OSR1. Voltage-gated K channel activity was quantified utilizing dual electrode voltage clamp and Kv1.5 channel protein abundance in the cell membrane utilizing chemiluminescence of Kv1.5 containing an extracellular hemagglutinin epitope (Kv1.5-HA). Kv1.5 activity and Kv1.5-HA protein abundance were significantly decreased by wild-type SPAK and SPAK, but not by SPAK and SPAK. Similarly, Kv1.5 activity and Kv1.5-HA protein abundance were significantly down-regulated by wild-type OSR1 and OSR1, but not by OSR1 and OSR1. Both, SPAK and OSR1 decrease cell membrane Kv1.5 protein abundance and activity. [ABSTRACT FROM AUTHOR]

Background/Aims: The transmembrane Klotho protein contributes to inhibition of 1,25(OH)2D3 formation. The extracellular domain of Klotho protein could function as an enzyme with e.g. β-glucuronidase activity, be cleaved off and be released into blood and cerebrospinal fluid. Klotho regulates several cellular transporters. Klotho protein deficiency accelerates the appearance of age related disorders including neurodegeneration and muscle wasting and eventually leads to premature death. The main site of Klotho protein expression is the kidney. Klotho protein is also appreciably expressed in other tissues including chorioid plexus. The present study explored the effect of Klotho protein on the creatine transporter CreaT (Slc6A8), which participates in the maintenance of neuronal function and survival. Methods: To this end cRNA encoding Slc6A8 was injected into Xenopus oocytes with and without additional injection of cRNA encoding Klotho protein. Creatine transporter CreaT (Slc6A8) activity was estimated from creatine induced current determined by two-electrode voltage-clamp. Results: Coexpression of Klotho protein significantly increased creatine-induced current in Slc6A8 expressing Xenopus oocytes. Coexpression of Klotho protein delayed the decline of creatine induced current following inhibition of carrier insertion into the cell membrane by brefeldin A (5 µM). The increase of creatine induced current by coexpression of Klotho protein in Slc6A8 expressing Xenopus oocytes was reversed by β-glucuronidase inhibitor (DSAL). Similarly, treatment of Slc6A8 expressing Xenopus oocytes with recombinant human alpha Klotho protein significantly increased creatine induced current. Conclusion: Klotho protein up-regulates the activity of creatine transporter CreaT (Slc6A8) by stabilizing the carrier protein in the cell membrane, an effect requiring β-glucuronidase activity of Klotho protein. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background/Aims: Transport regulation involves several kinases including SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1), which are under control of WNK (with-no-K[Lys]) kinases. The present study explored whether SPAK and/or OSR1 participate in the regulation of the creatine transporter CreaT (SLC6A8), which accomplishes Na+ coupled cellular uptake of creatine in several tissues including kidney, intestine, heart, skeletal muscle and brain. Methods: cRNA encoding SLC6A8 was injected into Xenopus laevis oocytes with or without additional injection of cRNA encoding wild-type SPAK, constitutively active T233ESPAK, WNK insensitive T233ASPAK, catalytically inactive D212ASPAK, wild-type OSR1, constitutively active T185EOSR1, WNK insensitive T185AOSR1 and catalytically inactive D164AOSR1. Transporter activity was determined from creatine (1 mM) induced current utilizing dual electrode voltage clamp. Results: Coexpression of wild-type SPAK and of T233ESPAK, but not of T233ASPAK or of D212ASPAK was followed by a significant decrease of creatine induced current in SLC6A8 expressing oocytes. Coexpression of SPAK significantly decreased maximal transport rate. Coexpression of wild-type OSR1, T185EOSR1 and T185AOSR1 but not of D164AOSR1 significantly negatively regulated SLC6A8 activity. OSR1 again decreased significantly maximal transport rate. Conclusions: Both, SPAK and OSR1, are negative regulators of the creatine transporter SLC6A8. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background/Aims: OSR1 (oxidative-stress-responsive kinase 1) participates in the regulation of renal tubular ion transport, cell volume and blood pressure. Whether OSR1 contributes to the regulation of organic solute transport remained; however, elusive. The present study thus explored the OSR1 sensitivity of the peptide transporters PEPT1 and PEPT2. Methods: cRNA encoding PEPT1 or PEPT2 were injected into Xenopus oocytes without or with additional injection of cRNA encoding wild-type OSR1, WNK1 insensitive inactive T185AOSR1, constitutively active T185EOSR1, and catalytically inactive D164AOSR1. Electrogenic peptide (glycine-glycine) transport was determined by dual electrode voltage clamp, the abundance of hemagglutinin-tagged PEPT2 (PEPT2-HA) by chemiluminescence. Results: In Xenopus oocytes injected with cRNA encoding PEPT1 or PEPT2, but not in oocytes injected with water, the dipeptide gly-gly (2 mM) generated an appreciable inward current (Igly-gly). Coexpression of OSR1 significantly decreased Igly-gly in both PEPT1 and PEPT2 expressing oocytes. The effect of OSR1 coexpression on Igly-gly in PEPT1 expressing oocytes was mimicked by coexpression of T185EOSR1, but not of D164AOSR1 or T185AOSR1. Kinetic analysis revealed that coexpression of OSR1 decreased maximal Igly-gly. OSR1 further decreased the PEPT2-HA protein abundance in the cell membrane. Conclusion: OSR1 has the capacity to downregulate the peptide transporters PEPT1 and PEPT2 by decreasing the carrier protein abundance in the cell membrane. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

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. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The WNK-dependent STE20/SPS1-related proline/alanine-rich kinase SPAK is a powerful regulator of ion transport. The study explored whether SPAK similarly regulates nutrient transporters, such as the Na-coupled glucose transporter SGLT1 (SLC5A1). To this end, SGLT1 was expressed in Xenopus oocytes with or without additional expression of wild-type SPAK, constitutively active SPAK, WNK-insensitive SPAK or catalytically inactive SPAK, and electrogenic glucose transport determined by dual-electrode voltage-clamp experiments. Moreover, Ussing chamber was employed to determine the electrogenic glucose transport in intestine from wild-type mice ( spak) and from gene-targeted mice carrying WNK-insensitive SPAK ( spak). In SGLT1-expressing oocytes, but not in water-injected oocytes, the glucose-dependent current ( I) was significantly decreased following coexpression of wild-type SPAK and SPAK, but not by coexpression of SPAK or SPAK. Kinetic analysis revealed that SPAK decreased maximal I without significantly modifying the glucose concentration required for halfmaximal I ( K). According to the chemiluminescence experiments, wild-type SPAK but not SPAK decreased SGLT1 protein abundance in the cell membrane. Inhibition of SGLT1 insertion by brefeldin A (5 μM) resulted in a decline of I, which was similar in the absence and presence of SPAK, suggesting that SPAK did not accelerate the retrieval of SGLT1 protein from the cell membrane but rather down-regulated carrier insertion into the cell membrane. Intestinal electrogenic glucose transport was significantly lower in spak than in spak mice. In conclusion, SPAK is a powerful negative regulator of SGLT1 protein abundance in the cell membrane and thus of electrogenic glucose transport. [ABSTRACT FROM AUTHOR]

Background/Aims: SPAK (STE20-related proline/alanine-rich kinase) is a powerful regulator of renal tubular ion transport and blood pressure. Moreover, SPAK contributes to the regulation of cell volume. Little is known, however, about a role of SPAK in the regulation or organic solutes. The present study thus addressed the influence of SPAK on the peptide transporters PEPT1 and PEPT2. Methods: To this end, cRNA encoding PEPT1 or PEPT2 were injected into Xenopus laevis oocytes without or with additional injection of cRNA encoding wild-type, SPAK, WNK1 insensitive inactive T233ASPAK, constitutively active T233ESPAK, and catalytically inactive D212ASPAK. Electrogenic peptide (glycine-glycine) transport was determined by dual electrode voltage clamp and PEPT2 protein abundance in the cell membrane by chemiluminescence. Intestinal electrogenic peptide transport was estimated from peptide induced current in Ussing chamber experiments of jejunal segments isolated from gene targeted mice expressing SPAK resistant to WNK-dependent activation (spaktg/tg) and respective wild-type mice (spak+/+). Results: In PEPT1 and in PEPT2 expressing oocytes, but not in oocytes injected with water, the dipeptide gly-gly (2 mM) generated an inward current, which was significantly decreased following coexpression of SPAK. The effect of SPAK on PEPT1 was mimicked by T233ESPAK, but not by D212ASPAK or T233ASPAK. SPAK decreased maximal peptide induced current of PEPT1. Moreover, SPAK decreased carrier protein abundance in the cell membrane of PEPT2 expressing oocytes. In intestinal segments gly-gly generated a current, which was significantly higher in spaktg/tg than in spak+/+ mice. Conclusion: SPAK is a powerful regulator of peptide transporters PEPT1 and PEPT2. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background/Aims: The kinases SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1) participate in the regulation of the NaCl cotransporter NCC and the Na+,K+,2Cl- cotransporter NKCC2. The kinases are regulated by WNK (with-no-K[Lys]) kinases. Mutations of genes encoding WNK kinases underly Gordon's syndrome, a monogenic disease leading to hypertension and hyperkalemia. WNK kinases further regulate the renal outer medullary K+ channel ROMK1. The present study explored, whether SPAK and/or OSR1 have similarly the potential to modify the activity of ROMK1. Methods: ROMK1 was expressed in Xenopus oocytes with or without additional expression of wild-type SPAK, constitutively active T233ESPAK, catalytically inactive D212ASPAK, wild-type OSR1, constitutively active T185EOSR1 and catalytically inactive D164AOSR1. Channel activity was determined utilizing dual electrode voltage clamp and ROMK1 protein abundance in the cell membrane utilizing chemiluminescence of ROMK1 containing an extracellular hemagglutinin epitope (ROMK1-HA). Results: ROMK1 activity and ROMK1-HA protein abundance were significantly down-regulated by wild-type SPAK and T233ESPAK, but not by D212ASPAK. Similarly, ROMK1 activity and ROMK1-HA protein abundance were significantly down-regulated by wild-type OSR1 and T185EOSR1, but not by D164AOSR1. Conclusion: ROMK1 protein abundance and activity are down-regulated by SPAK and OSR1. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background/Aims: SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1) are powerful regulators of diverse transport processes. Both kinases are activated by cell shrinkage and participate in stimulation of regulatory cell volume increase (RVI). Execution of RVI involves inhibition of Cl- channels. The present study explored whether SPAK and/or OSR1 regulate the activity of the Cl- channel ClC-2. Methods: To this end, ClC-2 was expressed in Xenopus laevis oocytes with or without additional expression of wild type SPAK, constitutively active SPAKT233E, WNK1 insensitive inactive SPAKT233A, catalytically inactive SPAKD212A, wild type OSR1, constitutively active OSR1T185E, WNK1 insensitive inactive OSR1T185A, and catalytically inactive OSR1D164A. Cl- channel activity was determined by dual electrode voltage clamp. Results: Expression of ClC-2 was followed by the appearance of a conductance (GCl), which was significantly decreased following coexpression of wild-type SPAK, SPAKT233E, wild type OSR1 or OSR1T185E, but not by coexpression of SPAKT233A, SPAKD212A, OSR1T185A, or OSR1D164A. Inhibition of ClC-2 insertion by brefeldin A (5 μM) resulted in a decline of GCl which was similar in the absence and presence of SPAK or OSR1, suggesting that SPAK and OSR1 did not accelerate the retrieval of ClC-2 protein from the cell membrane. Conclusion: SPAK and OSR1 are powerful negative regulators of the cell volume regulatory Cl- channel ClC-2. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Janus kinase 3 (JAK3) contributes to cytokine receptor signaling, confers cell survival and stimulates cell proliferation. The gain of function mutation JAK3 is found in acute megakaryoplastic leukemia. Replacement of ATP coordinating lysine by alanine yields inactive JAK3. Most recent observations revealed the capacity of JAK3 to regulate ion transport. This study thus explored whether JAK3 regulates glutamate transporters EAAT1-4, carriers accomplishing transport of glutamate and aspartate in a variety of cells including intestinal cells, renal cells, glial cells, and neurons. To this end, EAAT1, 2, 3, or 4 were expressed in Xenopus oocytes with or without additional expression of mouse wild-type JAK3, constitutively active JAK3 or inactive JAK3, and electrogenic glutamate transport was determined by dual electrode voltage clamp. Moreover, Ussing chamber was employed to determine electrogenic glutamate transport in intestine from mice lacking functional JAK3 ( jak3) and from corresponding wild-type mice ( jak3). As a result, in EAAT1, 2, 3, or 4 expressing oocytes, but not in oocytes injected with water, addition of glutamate to extracellular bath generated an inward current ( I), which was significantly increased following coexpression of JAK3. I in oocytes expressing EAAT3 was further increased by JAK3 but not by JAK3. I in EAAT3 + JAK3 expressing oocytes was significantly decreased by JAK3 inhibitor WHI-P154 (22 µM). Kinetic analysis revealed that JAK3 increased maximal I and significantly reduced the glutamate concentration required for half maximal I ( K). Intestinal electrogenic glutamate transport was significantly lower in jak3 than in jak3 mice. In conclusion, JAK3 is a powerful regulator of excitatory amino acid transporter isoforms. [ABSTRACT FROM AUTHOR]

Janus kinase-3 (JAK3) fosters proliferation and counteracts apoptosis of lymphocytes and tumor cells. The gain of function mutation JAK3 has been discovered in acute megakaryoplastic leukemia. JAK3 is inactivated by replacement of lysine by alanine in the catalytic subunit (JAK3). Regulation of cell proliferation and apoptosis involves altered activity of Cl channels. The present study, thus, explored whether JAK3 modifies the function of the small conductance Cl channel ClC-2. To this end, ClC-2 was expressed in Xenopus oocytes with or without wild-type JAK3, JAK3 or JAK3, and the Cl channel activity determined by dual-electrode voltage clamp. Channel protein abundance in the cell membrane was determined utilizing chemiluminescence. As a result, expression of ClC-2 was followed by a marked increase of cell membrane conductance. The conductance was significantly decreased following coexpression of JAK3 or JAK3, but not by coexpression of JAK3. Exposure of the oocytes expressing ClC-2 together with JAK3 to the JAK3 inhibitor WHI-P154 (4-[(3'-bromo-4'-hydroxyphenyl)amino]-6,7-dimethoxyquinazoline, 22 μM) increased the conductance. Coexpression of JAK3 decreased the ClC-2 protein abundance in the cell membrane of ClC-2 expressing oocytes. The decline of conductance in ClC-2 and JAK3 coexpressing oocytes following inhibition of channel protein insertion by brefeldin A (5 μM) was similar in oocytes expressing ClC-2 with JAK3 and oocytes expressing ClC-2 alone, indicating that JAK3 might slow channel protein insertion into rather than accelerating channel protein retrieval from the cell membrane. In conclusion, JAK3 downregulates ClC-2 activity and thus counteracts Cl exit-an effect possibly influencing cell proliferation and apoptosis. [ABSTRACT FROM AUTHOR]

Background/Aims: Renal tissue fibrosis contributes to the development of end-stage renal disease. Causes for renal tissue fibrosis include obstructive nephropathy. The development of renal fibrosis following unilateral ureteral obstruction (UUO) is blunted in gene-targeted mice lacking functional serum- and glucocorticoid-inducible kinase SGK1. Similar to Akt isoforms, SGK1 phosphorylates and thus inactivates glycogen synthase kinase GSK-3. The present study explored whether PKB/SGK-dependent phoshorylation of GSK-3α/β impacts on pro-fibrotic signaling following UUO. Methods: UUO was induced in mice carrying a PKB/SGK-resistant GSK-3α/β (gsk-3KI) and corresponding wild-type mice (gsk-3WT). Three days after the obstructive injury, expression of fibrosis markers in kidney tissues was analyzed by quantitative RT-PCR and western blotting. Results: GSK-3α and GSK-3β phosphorylation was absent in both, the non-obstructed and the obstructed kidney tissues from gsk-3KI mice but was increased by UUO in kidney tissues from gsk-3WT mice. Expression of α-smooth muscle actin, type I collagen and type III collagen in the non-obstructed kidney tissues was not significantly different between gsk-3KI mice and gsk-3WT mice but was significantly less increased in the obstructed kidney tissues from gsk-3KI mice than from gsk-3WT mice. After UUO treatment, renal β-catenin protein abundance and renal expression of the β-catenin sensitive genes: c-Myc, Dkk1, Twist and Lef1 were again significantly less increased in kidney tissues from gsk-3KI mice than from gsk-3WT mice. Conclusions: PKB/SGK-dependent phosphorylation of glycogen synthase kinase GSK-3 contributes to the pro-fibrotic signaling leading to renal tissue fibrosis in obstructive nephropathy. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background/Aims: The serum & glucocorticoid inducible kinase SGK3, an ubiquitously expressed serine/threonine kinase, regulates a variety of ion channels. It has previously been shown that SGK3 upregulates the outwardly rectifying K+ channel KV11.1, which is expressed in cardiomyocytes. Cardiomyocytes further express the inward rectifier K+ channel Kir2.1, which contributes to maintenance of resting cell membrane potential. Loss-of-function mutations of KCNJ2 encoding Kir2.1 result in Andersen-Tawil syndrome with periodic paralysis, cardiac arrhythmia and dysmorphic features. The present study explored whether SGK3 participates in the regulation of Kir2.1. Methods: cRNA encoding Kir2.1 was injected into Xenopus oocytes with and without additional injection of cRNA encoding wild type SGK3, constitutively active S419DSGK3 or inactive K191NSGK3. Kir2.1 activity was determined by two-electrode voltage-clamp and Kir2.1 protein abundance in the cell membrane by immunostaining and subsequent confocal imaging or by chemiluminescence. Results: Injection of 10 ng cRNA encoding wild type SGK3 and S419DSGK3, but not K191NSGK3 significantly enhanced Kir2.1-mediated currents. SGK inhibitor EMD638683 (50 µM) abrogated S419DSGK3-induced up-regulation of Kir2.1. Moreover, wild type SGK3 enhanced the channel protein abundance in the cell membrane. The decay of Kir2.1-mediated currents following inhibition of channel insertion into the cell membrane by brefeldin A (5 µM) was similar in oocytes coexpressing Kir2.1 and SGK3 as in oocytes expressing Kir2.1 alone, suggesting that SGK3 influences channel insertion into rather than channel retrieval from the cell membrane. Conclusions: SGK3 is a novel regulator of Kir2.1. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Besides their role in cardiac repolarization, human ether-a-go-go-related gene potassium (hERG) channels are expressed in several tumor cells including rhabdomyosarcoma cells. The channels foster cell proliferation. Ubiquitously expressed AMP-dependent protein kinase (AMPK) is a serine-/threonine kinase, stimulating energy-generating and inhibiting energy-consuming processes thereby helping cells survive periods of energy depletion. AMPK has previously been shown to regulate Na/K ATPase, Na/Ca exchangers, Ca channels and K channels. The present study tested whether AMPK regulates hERG channel activity. Wild type AMPK (α1β1γ1), constitutively active AMPK (α1β1γ1(R70Q)), or catalytically inactive AMPK (α1(K45R)β1γ1) were expressed in Xenopus oocytes with hERG. Tail currents were determined as a measure of hERG channel activity by two-electrode-voltage clamp. hERG membrane abundance was quantified by chemiluminescence and visualized by immunocytochemistry and confocal microscopy. Moreover, hERG currents were measured in RD rhabdomyosarcoma cells after pharmacological modification of AMPK activity using the patch clamp technique. Coexpression of wild-type AMPK and of constitutively active AMPK significantly downregulated the tail currents in hERG-expressing Xenopus oocytes. Pharmacological activation of AMPK with AICAR or with phenformin inhibited hERG currents in Xenopus oocytes, an effect abrogated by AMPK inhibitor compound C. AMPK enhanced the Nedd4-2-dependent downregulation of hERG currents. Coexpression of constitutively active AMPK decreased membrane expression of hERG in Xenopus oocytes. Compound C significantly enhanced whereas AICAR tended to inhibit hERG currents in RD rhabdomyosarcoma cells. AMPK is a powerful regulator of hERG-mediated currents in both, Xenopus oocytes and RD rhabdomyosarcoma cells. AMPK-dependent regulation of hERG may be particularly relevant in cardiac hypertrophy and tumor growth. [ABSTRACT FROM AUTHOR]

Highlights: [•] Coexpression of klotho enhances activity of cardiac K+-channel hERG. [•] Coexpression of klotho increases hERG protein abundance in the cell membrane. [•] The effect of expressed klotho is mimicked by recombinant klotho protein. [•] The effect of klotho is blunted by d-saccharic acid-1,4-lactone. [ABSTRACT FROM AUTHOR]

Background/Aims: Human parvovirus B19 (B19V) may cause inflammatory cardiomyopathy (iCMP) which is accompanied by endothelial dysfunction. The B19V capsid protein VP1 contains a lysophosphatidylcholine producing phospholipase A2 (PLA) sequence. Lysophosphatidylcholine has in turn been shown to inhibit Na+/K+ ATPase. The present study explored whether VP1 modifies Na+/K+ ATPase activity. Methods: Xenopus oocytes were injected with cRNA encoding VP1 isolated from a patient suffering from fatal B19V-iCMP or cRNA encoding PLA2-negative VP1 mutant (H153A) and K+ induced pump current (Ipump) as well as ouabain-inhibited current (Iouabain) both reflecting Na+/K+-ATPase activity were determined by dual electrode voltage clamp. Results: Injection of cRNA encoding VP1, but not of VP1(H153A) or water, was followed by a significant decrease of both, Ipump and Iouabain in Xenopus oocytes. The effect was not modified by inhibition of transcription with actinomycin (10 µM for 36 hours) but was abrogated in the presence of PLA2 specific blocker 4-bromophenacylbromide (50 µM) and was mimicked by lysophosphatidylcholine (0.5 - 1 µg/ml). According to whole cell patch clamp, lysophosphatidylcholine (1 µg /ml) similarly decreased Ipump in human microvascular endothelial cells (HMEC). Conclusion: The B19V capsid protein VP1 is a powerful inhibitor of host cell Na+/K+ ATPase, an effect at least partially due to phospholipase A2 (PLA2) dependent formation of lysophosphatidylcholine. Copyright © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]