PTPN2 Regulates the Interferon Signaling and Endoplasmic Reticulum Stress Response in Pancreatic ß-Cells in Autoimmune Diabetes.

Type 1 diabetes (T1D) results from autoimmune destruction of ß-cells in the pancreas. Protein tyrosine phosphatases (PTPs) are candidate genes for T1D and play a key role in autoimmune disease development and ß-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 ß-cells, we generated PTPN2-deficient human stem cell-derived ß-like and EndoC-ßH1 cells. Mechanistically, we demonstrated that PTPN2 inactivation in ß-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 ß-cells. Adenovirus-induced overexpression of PTPN2 partially protected from ER stress-induced ß-cell death. Our results postulate PTPN2 as a key protective factor in ß-cells during inflammation and ER stress in autoimmune diabetes.

STAT3 Regulates Mitochondrial Gene Expression in Pancreatic ß-Cells and Its Deficiency Induces Glucose Intolerance in Obesity.

Most obese and insulin-resistant individuals do not develop diabetes. This is the result of the capacity of ß-cells to adapt and produce enough insulin to cover the needs of the organism. The underlying mechanism of ß-cell adaptation in obesity, however, remains unclear. Previous studies have suggested a role for STAT3 in mediating ß-cell development and human glucose homeostasis, but little is known about STAT3 in ß-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 ß-cells, we generated mice with tamoxifen-inducible partial or full deletion of STAT3 in ß-cells and fed them a high-fat diet before analysis. Interestingly, ß-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-ß1H cells, confirmed in FACS-purified ß-cells from obese STAT3-deficient mice. Moreover, silencing of STAT3 impaired mitochondria activity in EndoC-ß1H cells and human islets, suggesting a mechanism for STAT3-modulated ß-cell function. Our study postulates STAT3 as a novel regulator of ß-cell function in obesity.

Sweet Killing in Obesity and Diabetes: The Metabolic Role of the BH3-only Protein BIM.

Diabetes is a metabolic disorder affecting more than 400 million individuals and their families worldwide. The major forms of diabetes (types 1 and 2) are characterized by pancreatic ß-cell dysfunction and, in some cases, loss of ß-cell mass causing hyperglycemia due to absolute or relative insulin deficiency. The BCL-2 homology 3 (BH3)-only protein BIM has a wide role in apoptosis induction in cells. In this review, we describe the apoptotic mechanisms mediated by BIM activation in ß cells in obesity and both forms of diabetes. We focus on molecular pathways triggered by inflammation, saturated fats, and high levels of glucose. Besides its role in cell death, BIM has been implicated in the regulation of mitochondrial oxidative phosphorylation and cellular metabolism in hepatocytes. BIM is both a key mediator of pancreatic ß-cell death and hepatic insulin resistance and is thus a potential therapeutic target for novel anti-diabetogenic drugs. We consider the implications and challenges of targeting BIM in the treatment of the disease.

Enhanced Reactive Oxygen Species Production, Acidic Cytosolic pH and Upregulated Na+/H+ Exchanger (NHE) in Dicer Deficient CD4+ T Cells.

BACKGROUND: MicroRNAs (miRNAs) negatively regulate gene expression at a post-transcriptional 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.

OSR1 and SPAK Sensitivity of Large-Conductance Ca2+ Activated K+ Channel.

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 (with-no-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+x0394;899-903 was injected into Xenopus laevis oocytes without or with additional injection of cRNA encoding wild-type OSR1 or wild-type SPAK, constitutively active T185EOSR1, 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+x0394;899-903 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.

Caveolin-1 Sensitivity of Excitatory Amino Acid Transporters EAAT1, EAAT2, EAAT3, and EAAT4.

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.

CD44 sensitivity of platelet activation, membrane scrambling and adhesion under high arterial shear rates.

CD44 is required for signalling of macrophage migration inhibitory factor (MIF), an anti-apoptotic pro-inflammatory cytokine. MIF is expressed and released from blood platelets, key players in the orchestration of occlusive vascular disease. Nothing is known about a role of CD44 in the regulation of platelet function. The present study thus explored whether CD44 modifies degranulation (P-selectin exposure), integrin activation, caspase activity, phosphatidylserine exposure on the platelet surface, platelet volume, Orai1 protein abundance and cytosolic Ca(2+)-activity ([Ca2+]i). Platelets from mice lacking CD44 (cd44(-/-)) were compared to platelets from corresponding wild-type mice (cd44(+/+)). In resting platelets, P-selectin abundance, α(IIb)ß3 integrin activation, caspase-3 activity and phosphatidylserine exposure were negligible in both genotypes and Orai1 protein abundance, [Ca2+]i, and volume were similar in cd44(-/-) and cd44(+/+) platelets. Platelet degranulation and α(IIb)ß3 integrin activation were significantly increased by thrombin (0.02 U/ml), collagen related peptide (CRP, 2 µg/ml and Ca(2+)-store depletion with thapsigargin (1 µM), effects more pronounced in cd44(-/-) than in cd44(+/+) platelets. Thrombin (0.02 U/ml) increased platelet [Ca2+]i, caspase-3 activity, phosphatidylserine exposure and Orai1 surface abundance, effects again significantly stronger in cd44(-/-) than in cd44(+/+) platelets. Thrombin further decreased forward scatter in cd44(-/-) and cd44(+/+) platelets, an effect which tended to be again more pronounced in cd44(-/-) than in cd44(+/+) platelets. Platelet adhesion and in vitro thrombus formation under high arterial shear rates (1,700 s(-1)) were significantly augmented in cd44(-/-) mice. In conclusion, genetic deficiency of CD44 augments activation, apoptosis and pro-thrombotic potential of platelets.

Up-Regulation of Voltage Gated K+ Channels Kv1.3 and Kv1.5 by Protein Kinase PKB/Akt.

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, HA-tagged 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 co-expression 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.

Regulation of Voltage Gated K+ Channel KCNE1/KCNQ1 by the Janus Kinase JAK3.

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.

SPAK and OSR1 Sensitive Kir2.1 K+ Channels.

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 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. 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 T233E SPAK, but not by T233A SPAK and D212A SPAK, as well as by wild-type OSR1 and T185E OSR1, but not by T185A OSR1 and D164A OSR1. 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.

SPAK and OSR1 Sensitive Cell Membrane Protein Abundance and Activity of KCNQ1/E1 K+ Channels.

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.

Up-Regulation of Intestinal Phosphate Transporter NaPi-IIb (SLC34A2) by the Kinases SPAK and OSR1.

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 (T233E)SPAK, WNK insensitive (T233A)SPAK, catalytically inactive (D212A)SPAK, wild-type OSR1, constitutively active (T185E)OSR1, WNK insensitive (T185A)OSR1 or catalytically inactive (D164A)OSR1. The phosphate (1 mM)-induced inward current (I(Pi)) was taken as measure of phosphate transport. RESULTS: I(Pi) was observed in NaPi-IIb expressing oocytes but not in water injected oocytes, and was significantly increased by co-expression of SPAK, (T233E)SPAK, OSR1, (T185E)OSR1 or SPAK+OSR1, but not by co-expression of (T233A)SPAK, (D212A)SPAK, (T185A)OSR1, or (D164A)OSR1. 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.

Down-Regulation of Excitatory Amino Acid Transporters EAAT1 and EAAT2 by the Kinases SPAK and OSR1.

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.

Regulation of Large Conductance Voltage-and Ca2+-Activated K+ Channels by the Janus Kinase JAK3.

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 Ca(2+)-activated K(+) channel BK. The present study explored whether JAK3 modifies BK channel protein abundance and current. METHODS: cRNA encoding Ca(2+)-insensitive BK channel (BK(M513I+Δ899-903)) was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild-type JAK3, constitutively active A568VJAK3, or inactive (K851A)JAK3. 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 BK(M513I+Δ899-903) expressing oocytes was slightly but significantly decreased by coexpression of wild-type JAK3 and of (A568V)JAK3, but not by coexpression of (K851A)JAK3. 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.

SPAK Sensitive Regulation of the Epithelial Na Channel ENaC.

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 (T233E)SPAK, WNK insensitive (T233A)SPAK or catalytically inactive (D212A)SPAK, 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 (spak(wt/wt)) and from gene targeted mice carrying WNK insensitive SPAK (spak(tg/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 (T233E)SPAK, but not following coexpression of (T233A)SPAK or (D212A)SPAK. Colonic Iamil was significantly higher in spak(wt/wt) than in spak(tg/tg) mice. CONCLUSION: SPAK has the potential to up-regulate ENaC.

SPAK and OSR1 Sensitivity of Excitatory Amino Acid Transporter EAAT3.

BACKGROUND/AIMS: Kinases involved in the regulation of epithelial transport include SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1). SPAK and OSR1 are both regulated by WNK (with-no-K(Lys)) kinases. The present study explored whether SPAK and/or OSR1 influence the excitatory amino acid transporter EAAT3, which accomplishes glutamate and aspartate transport in kidney, intestine and brain. METHODS: cRNA encoding EAAT3 was injected into Xenopus laevis 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. Glutamate-induced current was taken as measure of electrogenic glutamate transport and was quantified utilizing dual electrode voltage clamp. Furthermore, Ussing chamber was employed to determine glutamate transport in the intestine from gene-targeted mice carrying WNK insensitive SPAK (spak(tg/tg)) and from corresponding wild-type mice (spak(+/+)). RESULTS: EAAT3 activity was significantly decreased by wild-type SPAK and (T233E)SPAK, but not by (T233A)SPAK and (D212A)SPAK. SPAK decreased maximal transport rate without affecting significantly affinity of the carrier. Similarly, EAAT3 activity was significantly downregulated by wild-type OSR1 and (T185E)OSR1, but not by (T185A)OSR1 and (D164A)OSR1. Again OSR1 decreased maximal transport rate without affecting significantly affinity of the carrier. Intestinal electrogenic glutamate transport was significantly lower in spak(+/+) than in spak(tg/tg) mice. CONCLUSION: Both, SPAK and OSR1 are negative regulators of EAAT3 activity.

USP18 Sensitivity of Peptide Transporters PEPT1 and PEPT2.

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.

Regulation of Voltage-Gated K+ Channel Kv1.5 by the Janus Kinase JAK3.

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.

Up-regulation of Kv1.3 channels by janus kinase 2.

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 (K882E)JAK2 mutant, or human gain-of-function (V617F)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 (V617F)JAK2 but not (K882E)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.

Leucine-rich repeat kinase 2-sensitive Na+/Ca2+ exchanger activity in dendritic cells.

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, Ca(2+)-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 Ca(2+) levels ([Ca(2+)]i) were determined utilizing Fura-2 fluorescence and whole cell currents to decipher electrogenic transport. The increase of [Ca(2+)]i following inhibition of sarcoendoplasmatic Ca(2+)-ATPase with thapsigargin (1 µM) in the absence of extracellular Ca(2+) (Ca(2+)-release) and the increase of [Ca(2+)]i following subsequent readdition of extracellular Ca(2+) (SOCE) were both significantly larger in Lrrk2(-/-) than in Lrrk2(+/+) DCs. The augmented increase of [Ca(2+)]i could have been due to impaired Ca(2+) extrusion by K(+)-independent (NCX) and/or K(+)-dependent (NCKX) Na(+)/Ca(2+)-exchanger activity, which was thus determined from the increase of [Ca(2+)]i, (Δ[Ca(2+)]i), and current following abrupt replacement of Na(+) containing (130 mM) and Ca(2+) free (0 mM) extracellular perfusate by Na(+) free (0 mM) and Ca(2+) containing (2 mM) extracellular perfusate. As a result, both slope and peak of Δ[Ca(2+)]i as well as Na(+)/Ca(2+) 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(+)/Ca(2+)-exchanger activity, and significantly augmented the increase of [Ca(2+)]i following Ca(2+)-release and SOCE. In conclusion, the present observations disclose a completely novel functional significance of LRRK2, i.e., the up-regulation of Na(+)/Ca(2+) exchanger transcription and activity leading to attenuation of Ca(2+)-signals in DCs.