Zohreh Hosseinzadeh

Upregulation of Peptide Transporters PEPT1 and PEPT2 by Janus Kinase JAK2

Background/Aims: Janus-activated kinase-2 JAK2 participates in the signaling of several hormones including growth hormone, fosters tumor growth and modifies the activity of several Na+ coupled nutrient transporters. Peptide uptake into intestinal and tumor cells is accomplished by electrogenic peptide transporters PEPT1 and PEPT2. The present study thus explored whether JAK2 contributes to the regulation of PEPT1 and PEPT2 activity. Methods: cRNA encoding either PEPT1 or PEPT2 was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild type JAK2, constitutively active V617FJAK2 or inactive K882EJAK2. The current created by the dipeptide glycine-glycine (Igly-gly) was determined by dual electrode voltage clamp and taken as measure for electrogenic peptide transport. Results: No appreciable Igly-gly was observed in water injected oocytes. In PEPT1 or PEPT2 expressing oocytes Igly-gly was significantly increased by additional coexpression of JAK2. As shown in PEPT1 expressing oocytes, Igly-gly without significantly modifying the concentration required for halfmaximal Igly-gly (KM). Following disruption of carrier insertion with brefeldin A (5 µM) Igly-gly declined similarly fast in Xenopus oocytes expressing PEPT1 with JAK2 and in Xenopus oocytes expressing PEPT1 alone. In oocytes expressing both, PEPT1 and V617FJAK2, Igly-gly was gradually decreased by JAK2 inhibitor AG490 (40 µM). According to Ussing chamber experiments pharmacological JAK2 inhibition similarly decreased Igly-gly in mouse intestine. Conclusion: Regulation of the peptide transporters PEPT and PEPT2 does involve the Janus-activated kinase-2 JAK2.

Upregulation of Na + ,Cl - -Coupled Betaine/ γ-Amino-Butyric Acid Transporter BGT1 by Tau Tubulin Kinase 2

Background/Aims: The serine/threonine kinase Tau-tubulin-kinase 2 (TTBK2) is expressed in various tissues including kidney, liver and brain. Loss of function mutations of TTBK2 lead to autosomal dominant spinocerebellar ataxia type 11 (SCA11). Cell survival is fostered by cellular accumulation of organic osmolytes. Carriers accomplishing cellular accumulation of organic osmolytes include the Na+, Cl--coupled betaine/γ-amino-butyric acid transporter BGT1. The present study explored whether TTBK2 participates in the regulation of BGT1 activity. Methods: Electrogenic transport of GABA was determined in Xenopus oocytes expressing BGT1 with or without wild-type TTBK2, truncated TTBK2[1-450] or kinase inactive mutants TTBK2- KD and TTBK2[1-450]-KD. Results: Coexpression of wild-type TTBK2, but not of TTBK2[1-450], TTBK2-KD or TTBK2[1-450]-KD, increased electrogenic GABA transport. Wildtype TTBK2 increased the maximal transport rate without significantly modifying affinity of the carrier. Coexpression of wild-type TTBK2 significantly delayed the decline of transport following inhibition of carrier insertion with brefeldin A, indicating that wild-type TTBK2 increased carrier stability in the cell membrane. Conclusion: Tau-tubulin-kinase 2 TTBK2 is a powerful stimulator of the osmolyte and GABA transporter BGT1.

Downregulation of ClC-2 by JAK2.

JAK2 (Janus kinase-2) is activated by cell shrinkage and may thus participate in cell volume regulation. Cell volume regulatory ion channels include the small conductance Cl-channels ClC-2. The present study thus explored whether JAK2 influences ClC-2 activity. To this end, ClC-2 was expressed in Xenopus oocytes with or without wild type JAK2, active V617FJAK2 or inactive K882EJAK2 and the Cl channel activity determined by dual electrode voltage clamp. Expression of ClC-2 was followed by a marked increase of cell membrane conductance. The conductance was significantly decreased following coexpression of JAK2 or V617FJAK2, but not by coexpression of K882EJAK2. Exposure of the oocytes expressing ClC-2 together with V617FJAK2 to the JAK2 inhibitor AG490 (40 µM) resulted in a gradual increase of the conductance. According to chemiluminescence JAK2 decreased the channel protein abundance in the cell membrane. The decline of conductance in ClC-2 and V617FJAK2 coexpressing oocytes following inhibition of channel protein insertion by brefeldin A (5 µM) was similar in oocytes expressing ClC-2 with V617FJAK2 and oocytes expressing ClC-2 alone, indicating that V617FJAK2 might slow channel protein insertion into rather than accelerating channel protein retrieval from the cell membrane. In conclusion, JAK2 down-regulates ClC-2 activity and thus counteracts Cl-exit, an effect which may impact on cell volume regulation.

Regulation of the glutamate transporters by JAK2.

The Janus-activated kinase-2 JAK2 is involved in the signaling of leptin and erythropoietin receptors and mediates neuroprotective effects of the hormones. In theory, JAK2 could be effective through modulation of the glutamate transporters, carriers accounting for the clearance of glutamate released during neurotransmission. The present study thus elucidated the effect of JAK2 on the glutamate transporters EAAT1, EAAT2, EAAT3 and EAAT4. To this end, cRNA encoding the carriers was injected into Xenopus oocytes with or without cRNA encoding JAK2 and glutamate transport was estimated from glutamate induced current (Iglu). Iglu was observed in Xenopus oocytes expressing EAAT1 or EAAT2 or EAAT3 or EAAT4, but not in water injected oocytes. Coexpression of JAK2 resulted in an increase of Iglu by 83% (EAAT1), 67% (EAAT2), 42% (EAAT3) and 126% (EAAT4). As shown for EAAT4 expressing Xenopus oocytes, the effect of JAK2 was mimicked by gain of function mutation V617FJAK2 but not by the inactive mutant K882EJAK2. Incubation with JAK2 inhibitor AG490 (40 µM) resulted in a gradual decrease of Iglu by 53%, 79% and 92% within 3, 6 and 24 hours. Confocal microscopy and chemiluminescence analysis revealed that JAK2 coexpression increased EAAT4 protein abundance in the cell membrane. Disruption of transcription did not appreciably modify the up-regulation of Iglu in EAAT4 expressing oocytes. The decay of Iglu following inhibition of carrier insertion with brefeldin A was similar in oocytes expressing EAAT4 + JAK2 and oocytes expressing EAAT4 alone, indicating that JAK2 did not appreciably affect carrier retrieval from the membrane. In conclusion, JAK2 is a novel powerful regulator of glutamate transporters and thus participates in the protection against excitotoxicity.

Regulation of the Voltage Gated K+ Channel Kv1.3 by Recombinant Human Klotho Protein

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.

Down-Regulation of the Na + -Coupled Phosphate Transporter NaPi-IIa by AMP- Activated Protein Kinase

Background/Aims: The Na⁺-coupled phosphate transporter NaPi-IIa is the main carrier accomplishing renal tubular phosphate reabsorption. It is driven by the electrochemical Na⁺ gradient across the apical cell membrane, which is maintained by Na⁺ extrusion across the basolateral cell membrane through the Na⁺/K⁺ ATPase. The operation of NaPi-IIa thus requires energy in order to avoid cellular Na⁺ accumulation and K⁺ loss with eventual decrease of cell membrane potential, Cl^- entry and cell swelling. Upon energy depletion, early inhibition of Na⁺-coupled transport processes may delay cell swelling and thus foster cell survival. Energy depletion is sensed by the AMP-activated protein kinase (AMPK), a serine/threonine kinase stimulating several cellular mechanisms increasing energy production and limiting energy utilization. The present study explored whether AMPK influences the activity of NAPi-IIa. Methods: cRNA encoding NAPi-IIa was injected into Xenopus oocytes with or without additional expression of wild-type AMPK (AMPK^α1-HA+AMPK^β1-Flag+AMPK^γ1-HA), of inactive AMPK^αK45R (AMPK^α1K45R+AMPK^β1-Flag+AMPK^γ1-HA) or of constitutively active AMPK^γR70Q (AMPK^α1-HA+AMPK^β1-Flag+AMPKγ1^R70Q). NaPi-IIa activity was estimated from phosphate-induced current in dual electrode voltage clamp experiments. Results: In NaPi-IIa-expressing, but not in water-injected Xenopus oocytes, the addition of phosphate (1 mM) to the extracellular bath solution generated a current (I_p), which was significantly decreased by coexpression of wild-type AMPK and of AMPK^γR70Q but not of AMPK^αK45R. The phosphate-induced current in NaPi-IIa- and AMPK-expressing Xenopus ooocytes was significantly increased by AMPK inhibitor Compound C (20 µM). Kinetic analysis revealed that AMPK significantly decreased the maximal transport rate. Conclusion: The AMP-activated protein kinase AMPK is a powerful regulator of NaPi-IIa and thus of renal tubular phosphate transport.

SPAK Dependent Regulation of Peptide Transporters PEPT1 and PEPT2

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.

Regulation of ClC-2 activity by SPAK and OSR1.

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 SPAK^T233E, WNK1 insensitive inactive SPAK^T233A, catalytically inactive SPAK^D212A, wild type OSR1, constitutively active OSR1^T185E, WNK1 insensitive inactive OSR1^T185A, and catalytically inactive OSR1^D164A. Cl^- channel activity was determined by dual electrode voltage clamp. Results: Expression of ClC-2 was followed by the appearance of a conductance (G_Cl), which was significantly decreased following coexpression of wild-type SPAK, SPAK^T233E, wild type OSR1 or OSR1^T185E, but not by coexpression of SPAK^T233A, SPAK^D212A, OSR1^T185A, or OSR1^D164A. Inhibition of ClC-2 insertion by brefeldin A (5 μM) resulted in a decline of G_Cl 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.

Downregulation of Peptide Transporters PEPT1 and PEPT2 by Oxidative Stress Responsive Kinase OSR1

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.

Stimulation of the glucose carrier SGLT1 by JAK2

JAK2 (Janus kinase-2) overactivity contributes to survival of tumor cells and the (V617F)JAK2 mutant is found in the majority of myeloproliferative diseases. Tumor cell survival depends on availability of glucose. Concentrative cellular glucose uptake is accomplished by Na(+) coupled glucose transport through SGLT1 (SLC5A1), which may operate against a chemical glucose gradient and may thus be effective even at low extracellular glucose concentrations. The present study thus explored whether JAK2 activates SGLT1. To this end, SGLT1 was expressed in Xenopus oocytes with or without wild type JAK2, (V617F)JAK2 or inactive (K882E)JAK2 and electrogenic glucose transport determined by dual electrode voltage clamp experiments. In SGLT1-expressing oocytes but not in oocytes injected with water or JAK2 alone, the addition of glucose to the extracellular bath generated a current (I(g)), which was significantly increased following coexpression of JAK2 or (V617F)JAK2, but not by coexpression of (K882E)JAK2. Kinetic analysis revealed that coexpression of JAK2 enhanced the maximal transport rate without significantly modifying the affinity of the carrier. The stimulating effect of JAK2 expression was abrogated by preincubation with the JAK2 inhibitor AG490. Chemiluminescence analysis revealed that JAK2 enhanced the carrier protein abundance in the cell membrane. The decline of I(g) during inhibition of carrier insertion by brefeldin A was similar in the absence and presence of JAK2. Thus, JAK2 fosters insertion rather than inhibiting retrieval of carrier protein into the cell membrane. In conclusion, JAK2 upregulates SGLT1 activity which may play a role in the effect of JAK2 during ischemia and malignancy.