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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]

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]

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]

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]

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]

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]

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]

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]