Bernat Elvira

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 Ca²⁺-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_llbβ₃ inte-grin 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_llbβ₃ integrin activation were significantly increased by thrombin (0.02 U/ml), collagen related peptide (CRP, 2 µg/ml and Ca²⁺-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 prothrombotic potential of platelets.

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.

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.

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.

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

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.

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

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 ^T233ESPAK, WNK insensitive ^T233ASPAK, catalytically inactive ^D212ASPAK, constitutively active ^T185EOSR1, WNK insensitive ^T185AOSR1 or catalytically inactive ^D164AOSR1. 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 ^T233ESPAK and ^T185EOSR1, but not by ^T233ASPAK, ^D212ASPAK, ^T185AOSR1, or ^D164AOSR1. 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.

Background/Aims: Janus kinase 3 (JAK3), a tyrosine kinase contributing to the regulation of cell proliferation and apoptosis of lymphocytes and tumour cells, has been shown to modify the expression and function of several ion channels and transport proteins. Channels involved in the regulation of cell proliferation include the large conductance voltage- and Ca²⁺-activated K⁺ channel BK. The present study explored whether JAK3 modifies BK channel protein abundance and current. Methods: cRNA encoding Ca²⁺-insensitive BK channel (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 ^K851AJAK3. Voltage gated K⁺ channel activity was measured utilizing dual electrode voltage clamp. Moreover, BK channel protein abundance was determined utilizing flow cytometry in CD19⁺ B lymphocyte cell membranes from mice lacking functional JAK3 (jak3^-/-) and corresponding wild-type mice (jak3^+/+). Results: BK activity in BK^{M513I+Δ899-903} expressing oocytes was slightly but significantly decreased by coexpression of wild-type JAK3 and of ^A568VJAK3, but not by coexpression of ^K851AJAK3. The BK channel protein abundance in the cell membrane was significantly higher in jak3^-/- than in jak3^+/+ B lymphocytes. The decline of conductance in BK and JAK3 coexpressing oocytes following inhibition of channel protein insertion by brefeldin A (5 µM) was similar in oocytes expressing BK with JAK3 and oocytes expressing BK alone, indicating that JAK3 might slow channel protein insertion into rather than accelerating channel protein retrieval from the cell membrane. Conclusion: JAK3 is a weak negative regulator of membrane BK protein abundance and activity.