Up-Regulation of Na+/Ca2+ Exchange in Megakaryocytes Following TGFß1 Treatment.

BACKGROUND: Blood platelets are activated by increase of cytosolic Ca2+ activity ([Ca2+]i). Ca2+ entry is accomplished in part by store operated Ca2+ entry (SOCE) involving Ca2+ release activated Ca2+-channel (CRAC) moiety Orai1 and its regulator STIM1, which are stimulated by depletion of intracellular Ca2+ stores. An increase of [Ca2+]i is terminated by Na+/Ca2+-exchange. The expression of both, Orai1 and STIM1 in megakaryocytes is up-regulated by tumor growth factor TGFß1, a powerful regulator of megakaryocyte differentiation. The present study explored whether TGFß1 similarly modifies megakaryocyte Na+/Ca2+-exchanger activity. METHODS: [Ca2+]i was determined utlizing Fura-2 fluorescence, SOCE from increase of [Ca2+]i, following readdition of extracellular Ca2+ after store depletion, and Na+/Ca2+-exchanger activity from increase of [Ca2+]i and whole cell currents following removal of extracellular Na+. RESULTS: TGFß1 treatment not only augments the increase of [Ca2+]i following store depletion and SOCE, but significantly up-regulates Na+/Ca2+-exchanger activity as apparent from [Ca2+]i measurements and whole cell currents. CONCLUSIONS: TGFß1 is a powerful stimulator of both, SOCE and Na+/Ca2+-exchanger activity in megakaryocytes.

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.

Effect of TGFß on calcium signaling in megakaryocytes.

TGFß is a powerful regulator of megakaryocyte maturation and platelet formation. As previously shown for other cell types, TGFß may up-regulate the expression of the serum & glucocorticoid inducible kinase SGK1, an effect requiring p38 kinase. SGK1 has in turn recently been shown to participate in the regulation of cytosolic Ca(2+) activity ([Ca(2+)]i) in megakaryocytes and platelets. SGK1 phosphorylates the IκB kinase (IKKα/ß), which in turn phosphorylates the inhibitor protein IκBα resulting in nuclear translocation of nuclear factor NFκB. Genes up-regulated by NFκB include Orai1, the pore forming ion channel subunit accomplishing store operated Ca(2+) entry (SOCE). The present study explored whether TGFß influences Ca(2+) signaling in megakaryocytes. [Ca(2+)]i was determined by Fura-2 fluorescence and SOCE from the increase of [Ca(2+)]i following re-addition of extracellular Ca(2+) after store depletion by removal of extracellular Ca(2+) and inhibition of the sarcoendoplasmatic Ca(2+) ATPase (SERCA) with thapsigargin (1 µM). As a result, TGFß (60 ng, 24 h) increased SOCE, an effect significantly blunted by p38 kinase inhibitor Skepinone-L (1 µM), SGK1 inhibitor EMD638683 (50 µM) and NFκB inhibitor wogonin (100 µM). In conclusion, TGFß is a powerful regulator of store operated Ca(2+) entry into megakaryocytes, an effect mediated by a signaling cascade involving p38 kinase, SGK1 and NFκB.

Up-regulation of megakaryocytic Na(+)/Ca(2+) exchange in klotho-deficient mice.

The active form of vitamin D, 1,25(OH)2D3, is a powerful regulator of cytosolic Ca(2+)-concentration ([Ca(2+)]i) in a variety of cell types. The formation of 1,25(OH)2D3 is inhibited by FGF23, an effect requiring presence of klotho. 1,25(OH)2D3 plasma levels are excessive in klotho-deficient mice (kl/kl). A previous study revealed that klotho-deficiency is followed by decreased activation of platelets, an effect at least in part due to blunted store operated Ca(2+) entry (SOCE). In other cell types 1,25(OH)2D3 has been shown to up-regulate the Na(+)/Ca(2+)-exchanger, which could, depending on cell membrane potential and cytosolic Na(+) concentration, either decrease or increase [Ca(2+)]i. The present study explored whether Na(+)/Ca(2+)-exchanger activity is different in megakaryocytes isolated from kl/kl mice than in megakaryocytes isolated from wild type mice. Na(+)/Ca(2+)-exchanger induced currents were determined by whole cell patch clamp and the Na(+)/Ca(2+)-exchanger induced alterations of [Ca(2+)]i by Fura-2 fluorescence. As a result, the inward current and the increase of [Ca(2+)]i following replacement of extracellular Na(+) by NMDG were higher in kl/kl megakaryocytes than in wild type megakaryocytes, a difference abrogated by treatment of the mice with low Vitamin D diet. Pretreatment of wild type megakaryocytes with 1,25(OH)2D3 (100 nM, 48 h) was followed by enhancement of both, inward current and increase of [Ca(2+)]i following replacement of extracellular Na(+) by NMDG. In conclusion, the present observations reveal a powerful stimulating effect of 1,25(OH)2D3 on Na(+)/Ca(2+)-exchanger activity in megakaryocytes.

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.

Down-regulation of inwardly rectifying Kir2.1 K+ channels by human parvovirus B19 capsid protein VP1.

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.

Upregulation of the large conductance voltage- and Ca2+-activated K+ channels by Janus kinase 2.

The iberiotoxin-sensitive large conductance voltage- and Ca(2+)-activated potassium (BK) channels (maxi-K(+)-channels) hyperpolarize the cell membrane thus supporting Ca(2+) entry through Ca(2+)-release activated Ca(2+) channels. Janus kinase-2 (JAK2) has been identified as novel regulator of ion transport. To explore whether JAK2 participates in the regulation of BK channels, cRNA encoding Ca(2+)-insensitive BK channels (BK(M513I+Δ899-903)) was injected into Xenopus oocytes with or without cRNA encoding wild-type JAK2, gain-of-function (V617F)JAK2, or inactive (K882E)JAK2. K(+) conductance was determined by dual electrode voltage clamp and BK-channel protein abundance by confocal microscopy. In A204 alveolar rhabdomyosarcoma cells, iberiotoxin-sensitive K(+) current was determined utilizing whole cell patch clamp. A204 cells were further transfected with JAK2 and BK-channel transcript, and protein abundance was quantified by RT-PCR and Western blotting, respectively. As a result, the K(+) current in BK(M513I+Δ899-903)-expressing oocytes was significantly increased following coexpression of JAK2 or (V617F)JAK2 but not (K882E)JAK2. Coexpression of the BK channel with (V617F)JAK2 but not (K882E)JAK2 enhanced BK-channel protein abundance in the oocyte cell membrane. Exposure of BK-channel and (V617F)JAK2-expressing oocytes to the JAK2 inhibitor AG490 (40 µM) significantly decreased K(+) current. Inhibition of channel insertion by brefeldin A (5 µM) decreased the K(+) current to a similar extent in oocytes expressing the BK channel alone and in oocytes expressing the BK channel and (V617F)JAK2. The iberiotoxin (50 nM)-sensitive K(+) current in rhabdomyosarcoma cells was significantly decreased by AG490 pretreatment (40 µM, 12 h). Moreover, overexpression of JAK2 in A204 cells significantly enhanced BK channel mRNA and protein abundance. In conclusion, JAK2 upregulates BK channels by increasing channel protein abundance in the cell membrane.

Up-regulation of Na(+)-coupled glucose transporter SGLT1 by caveolin-1.

The Na(+)-coupled glucose transporter SGLT1 (SLC5A1) accomplishes concentrative cellular glucose uptake even at low extracellular glucose concentrations. The carrier is expressed in renal proximal tubules, small intestine and a variety of nonpolarized cells including several tumor cells. The present study explored whether SGLT1 activity is regulated by caveolin-1, which is known to regulate the insertion of several ion channels and carriers in the cell membrane. To this end, SGLT1 was expressed in Xenopus oocytes with or without additional expression of caveolin-1 and electrogenic glucose transport determined by dual electrode voltage clamp experiments. In SGLT1-expressing oocytes, but not in oocytes injected with water or caveolin-1 alone, the addition of glucose to the extracellular bath generated an inward current (Ig), which was increased following coexpression of caveolin-1. Kinetic analysis revealed that caveolin-1 increased maximal Ig without significantly modifying the glucose concentration required to trigger half maximal Ig (KM). According to chemiluminescence and confocal microscopy, caveolin-1 increased SGLT1 protein abundance in the cell membrane. Inhibition of SGLT1 insertion by brefeldin A (5µM) resulted in a decline of Ig, which was similar in the absence and presence of caveolin-1. In conclusion, caveolin-1 up-regulates SGLT1 activity by increasing carrier protein abundance in the cell membrane, an effect presumably due to stimulation of carrier protein insertion into the cell membrane.

Up-regulation of Kir2.1 (KCNJ2) by the serum & glucocorticoid inducible SGK3.

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 K(ir)2.1, which contributes to maintenance of resting cell membrane potential. Loss-of-function mutations of KCNJ2 encoding K(ir)2.1 result in Andersen-Tawil syndrome with periodic paralysis, cardiac arrhythmia and dysmorphic features. The present study explored whether SGK3 participates in the regulation of K(ir)2.1. METHODS: cRNA encoding K(ir)2.1 was injected into Xenopus oocytes with and without additional injection of cRNA encoding wild type SGK3, constitutively active (S419D)SGK3 or inactive (K191N)SGK3. Kir2.1 activity was determined by two-electrode voltage-clamp and K(ir)2.1 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 (S419D)SGK3, but not (K191N)SGK3 significantly enhanced K(ir)2.1-mediated currents. SGK inhibitor EMD638683 (50 µM) abrogated (S419D)SGK3-induced up-regulation of K(ir)2.1. Moreover, wild type SGK3 enhanced the channel protein abundance in the cell membrane. The decay of K(ir)2.1-mediated currents following inhibition of channel insertion into the cell membrane by brefeldin A (5 µM) was similar in oocytes coexpressing K(ir)2.1 and SGK3 as in oocytes expressing K(ir)2.1 alone, suggesting that SGK3 influences channel insertion into rather than channel retrieval from the cell membrane. CONCLUSIONS: SGK3 is a novel regulator of K(ir)2.1.

Akt2- and ETS1-dependent IP3 receptor 2 expression in dendritic cell migration.

BACKGROUND/AIMS: The protein kinase Akt2/PKBß is a known regulator of macrophage and dendritic cell (DC) migration. The mechanisms linking Akt2 activity to migration remained, however, elusive. DC migration is governed by Ca(2+) signaling. We thus explored whether Akt2 regulates DC Ca(2+) signaling. METHODS: DCs were derived from bone marrow of Akt2-deficient mice (akt2(-/-)) and their wild type littermates (akt2(+/+)). DC maturation was induced by lipopolysaccharides (LPS) and evaluated by flow cytometry. Cytosolic Ca(2+) concentration was determined by Fura-2 fluorescence, channel activity by whole cell recording, transcript levels by RT-PCR, migration utilizing transwells. RESULTS: Upon maturation, chemokine CCL21 stimulated migration of akt2(+/+) but not akt2(-/-) DCs. CCL21-induced increase in cytosolic Ca(2+) concentration, thapsigargin-induced release of Ca(2+) from intracellular stores with subsequent store-operated Ca(2+) entry (SOCE), ATP-induced inositol 1,4,5-trisphosphate (IP3)-dependent Ca(2+) release as well as Ca(2+) release-activated Ca(2+) (CRAC) channel activity were all significantly lower in mature akt2(-/-) than in mature akt2(+/+) DCs. Transcript levels of IP3 receptor IP3R2 and of IP3R2 regulating transcription factor ETS1 were significantly higher in akt2(+/+) than in akt2(-/-) DCs prior to maturation and were upregulated by LPS stimulation (1h) in akt2(+/+) and to a lower extent in akt2(-/-) DCs. Following maturation, protein abundance of IP3R2 and ETS1 were similarly higher in akt2(+/+) than in akt2(-/-) DCs. The IP3R inhibitor Xestospongin C significantly decreased CCL21-induced migration of akt2(+/+)DCs and abrogated the differences between genotypes. Finally, knock-down of ETS1 with siRNA decreased IP3R2 mRNA abundance, thapsigargin- and ATP-induced Ca(2+) release, SOCE and CRAC channel activation, as well as DC migration. CONCLUSION: Akt2 upregulates DC migration at least in part by ETS1-dependent stimulation of IP3R2 transcription.

Down-regulation of K⁺ channels by human parvovirus B19 capsid protein VP1.

Parvovirus B19 (B19V) can cause inflammatory cardiomyopathy and endothelial dysfunction. Pathophysiological mechanisms involved include lysophosphatidylcholine producing phospholipase A2 (PLA2) activity of the B19V capsid protein VP1. Most recently, VP1 and lysophosphatidylcholine have been shown to inhibit Na(+)/K(+) ATPase. The present study explored whether VP1 modifies the activity of Kv1.3 and Kv1.5 K(+) channels. cRNA encoding Kv1.3 or Kv1.5 was injected into Xenopus oocytes without or with cRNA encoding VP1 isolated from a patient suffering from fatal B19V-induced myocarditis. K(+) channel activity was determined by dual electrode voltage clamp. Injection of cRNA encoding Kv1.3 or Kv1.5 into Xenopus oocytes was followed by appearance of Kv K(+) channel activity, which was significantly decreased by additional injection of cRNA encoding VP1, but not by additional injection of cRNA encoding PLA2-negative VP1 mutant (H153A). The effect of VP1 on Kv current was not significantly modified by transcription inhibitor actinomycin (10 µM for 36 h) but was mimicked by lysophosphatidylcholine (1 µg/ml). The B19V capsid protein VP1 inhibits host cell Kv channels, an effect at least partially due to phospholipase A2 (PLA) dependent formation of lysophosphatidylcholine.

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 ß-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 ß-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 ß-glucuronidase activity.

Upregulation of the creatine transporter Slc6A8 by Klotho.

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. ß-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 ß-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 ß-glucuronidase activity of Klotho protein.

AMP-activated protein kinase regulates hERG potassium channel.

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⁺/Ca²âº exchangers, Ca²âº channels and K⁺ channels. The present study tested whether AMPK regulates hERG channel activity. Wild type AMPK (α1ß1γ1), constitutively active (γR70Q)AMPK (α1ß1γ1(R70Q)), or catalytically inactive (αK45R)AMPK (α1(K45R)ß1γ1) 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 (γR70Q)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. (γR70Q)AMPK enhanced the Nedd4-2-dependent downregulation of hERG currents. Coexpression of constitutively active (γR70Q)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.

Downregulation of the renal outer medullary K(+) channel ROMK by the AMP-activated protein kinase.

The 5'-adenosine monophosphate-activated serine/threonine protein kinase (AMPK) is stimulated by energy depletion, increase in cytosolic Ca(2+) activity, oxidative stress, and nitric oxide. AMPK participates in the regulation of the epithelial Na(+) channel ENaC and the voltage-gated K(+) channel KCNE1/KCNQ1. It is partially effective by decreasing PIP(2) formation through the PI3K pathway. The present study explored whether AMPK regulates the renal outer medullary K(+) channel ROMK. To this end, cRNA encoding ROMK was injected into Xenopus oocytes with and without additional injection of constitutively active AMPK(γR70Q) (AMPK(α1)-HA+AMPK(ß1)-Flag+AMPKγ1(R70Q)), or of inactive AMPK(αK45R) (AMPK(α1K45R)+AMPK(ß1)-Flag+AMPK(γ1)-HA), and the current determined utilizing two-electrode voltage-clamp and single channel patch clamp. ROMK protein abundance was measured utilizing chemiluminescence in Xenopus oocytes and western blot in whole kidney tissue. Moreover, renal Na(+) and K(+) excretion were determined in AMPK(α1)-deficient mice (ampk ( -/- )) and wild-type mice (ampk ( +/+ )) prior to and following an acute K(+) load (111 mM KCl, 30 mM NaHCO(3), 4.7 mM NaCl, and 2.25 g/dl BSA) at a rate of 500 µl/h. As a result, coexpression of AMPK(γR70Q) but not of AMPK(αK45R) significantly decreased the current in ROMK1-expressing Xenopus oocytes. Injection of phosphatidylinositol PI((4,5))P(2) significantly increased the current in ROMK1-expressing Xenopus oocytes, an effect reversed in the presence of AMPK(γR70Q). Under control conditions, no significant differences between ampk ( -/- ) and ampk ( +/+ ) mice were observed in glomerular filtration rate (GFR), urinary flow rate, serum aldosterone, plasma Na(+), and K(+) concentrations as well as absolute and fractional Na(+) and K(+) excretion. Following an acute K(+) load, GFR, urinary flow rate, serum aldosterone, plasma Na(+), and K(+) concentration were again similar in both genotypes, but renal absolute and fractional Na(+) and K(+) excretion were higher in ampk ( -/- ) than in ampk ( +/+ ) mice. According to micropuncture following a K(+) load, delivery of Na(+) to the early distal tubule but not delivery of K(+) to late proximal and early distal tubules was increased in ampk (-/-) mice. The upregulation of renal ROMK1 protein expression by acute K(+) load was more pronounced in ampk (-/-) than in ampk ( +/+ ) mice. In conclusion, AMPK downregulates ROMK, an effect compromising the ability of the kidney to excrete K(+) following an acute K(+) load.

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.

PIKfyve sensitivity of hERG channels.

BACKGROUND/AIMS: Human ether-a-go-go (hERG) channels contribute to cardiac repolarization and participate in the regulation of tumor cell proliferation. Mutations in hERG channels may cause long QT syndrome and sudden cardiac death due to ventricular arrhythmias. HERG channel activity is up-regulated by the serum- and glucocorticoid-inducible kinase isoforms SGK1 and SGK3. Related kinases are protein kinase B (PKB/Akt) isoforms. SGK´s and PKB/Akt´s activate phosphatidylinositol-3-phosphate-5-kinase PIKfyve, which in turn up-regulates several carriers and channels. An effect of PIKfyve on hERG channels, has, however, never been shown. The present study thus explored the putative influence of PIKfyve on hERG channel expression and activity. METHODS: hERG channels were expressed in Xenopus oocytes with or without PIKfyve and/or PKB, expression of endogenous and injected hERG quantified by RT-PCR, and hERG channel activity determined utilizing dual electrode voltage clamp. Moreover, hERG protein abundance in the cell membrane was visualized utilizing specific antibody binding and subsequent confocal microscopy and quantified by chemiluminescence. RESULTS: Coexpression of wild type PIKfyve increased hERG channel activity in hERG-expressing Xenopus oocytes. hERG channel activity was further increased by coexpression of PKB, an effect augmented by additional coexpression of PIKfyve, but not by additional coexpression of PKB/Akt-resistant PIKfyve mutant PIKfyve(S318A). Coexpression of PIKfyve increased hERG channel protein abundance in the cell membrane. Inhibition of hERG channel insertion into the cell membrane by Brefeldin A (5 µM) resulted in a decline of current, which was similar in Xenopus oocytes expressing hERG together with PIKfyve and in Xenopus oocytes expressing hERG alone. CONCLUSION: hERG is up-regulated by PIKfyve, which is in turn activated by PKB/Akt.

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 (V617F)JAK2 or inactive (K882E)JAK2. 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 (V617F)JAK2, 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.

Down-regulation of Na/K+ atpase activity by human parvovirus B19 capsid protein VP1.

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 (I(pump)) as well as ouabain-inhibited current (I(ouabain)) 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, I(pump) and I(ouabain) 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 I(pump) 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.

Up-regulation of the inwardly rectifying K⁺ channel Kir2.1 (KCNJ2) by protein kinase B (PKB/Akt) and PIKfyve.

The inward rectifier K⁺ channel Kir2.1 contributes to the maintenance of the resting cell membrane potential in excitable cells. Loss of function mutations of KCNJ2 encoding Kir2.1 result in Andersen-Tawil syndrome, a disorder characterized by periodic paralysis, cardiac arrhythmia, and dysmorphic features. The ubiquitously expressed protein kinase B (PKB/Akt) activates the phosphatidylinositol-3-phosphate-5-kinase PIKfyve, which in turn regulates a variety of carriers and channels. The present study explored whether PKB/PIKfve contributes to the regulation of Kir2.1. To this end, cRNA encoding Kir2.1 was injected into Xenopus oocytes with and without additional injection of cRNA encoding wild type PKB (PKB), constitutively active (T308D,S473D)PKB or inactive (T308A,S473A)PKB. Kir2.1 activity was determined by two-electrode voltage-clamp. As a result, PKB and (T308D,S473D)PKB, but not (T308A,S473A)PKB, significantly increased Kir2.1-mediated currents. The effect of PKB was mimicked by coexpression of PIKfyve but not of (S318A)Pikfyve lacking the PKB phosphorylation site. The decay of Kir2.1-mediated currents after inhibition of channel insertion into the cell membrane by brefeldin A (5 µM) was similar in oocytes expressing Kir2.1 + PKB or Kir2.1 + PIKfyve to those expressing Kir2.1 alone, suggesting that PKB and PIKfyve influence channel insertion into rather than channel retrieval from the cell membrane. In conclusion, PKB and PIKfyve are novel regulators of Kir2.1.