Localization of the Plasmodium Falciparum K+ Channel, Pfkch1
1
Our results point to KCa1.1 channels as a promising new target for RA therapy and open the possibility for treatments that would not induce immunosuppression. 2787-Pos Board B479 The Functional Swtich in Potassium Channels in Myotonic Dystrophy Type 1 Impairs Proliferation, Migration and Fusion During Myogenesis Rajeev B. Tajhya, Xueyou Hu, Mark R. Tanner, Lubov Timchenko, Christine Beeton. Baylor College of Medicine, Houston, TX, USA. Voltage-gated K þ channels (Kv) are responsible for myoblasts proliferation and differentiation by triggering changes in membrane potential and cell volume. Since individuals with myotonic dystrophy type 1 (DM1) display reduced myogenesis led by prolonged myoblasts proliferation and delayed myotubes fusion, we investigated the roles of K þ channels in primary human myoblasts obtained from DM1 patients and healthy volunteers. DM1 is an autosomal dominant neuromuscular disorder affecting 1 in 8000 people world- wide. It is the most common adult-onset muscular dystrophy and currently has no treatment. DM1 is characterized by muscle wasting and multi-system disorders. We have identified a switch in functional potassium channel expression from KCa1.1 to Kv1 channels when comparing myoblasts from healthy individuals to myoblasts from patients with DM1. We showed increase in Kv1.2 and Kv1.5 channels, and decrease in KCa1.1 channels in DM1 myoblasts at mRNA level by RT-PCR, at protein level by immunofluorescence, and at channel activity by patch-clamp technique. We hypothesized that this switch in K þ channels plays a role in the reduced myogenesis observed in patients with DM1, and that selective inhibition of Kv1 channels rescues the pathological features of DM1 in skeletal muscle. We show that pharmacological inhibition of Kv1 channels in DM1 myoblasts normalized proliferation, rescued matrix metalloproteinase-2 (MMP-2, a protease necessary for myotube fusion) production, and partially rescued myotube fusion shown as increase in fusion index. On the contrary, selective inhibition of KCa1.1 in normal myoblasts lowered MMP-2 production, impaired wound healing repair, and decreased myotubes formation. Therefore we conclude that loss of KCa1.1 and up-regulation of Kv1 channels in DM1 impairs early stage of myogenesis and can be partially rescued by modulating such K þ channels. 2788-Pos Board B480 Altered Gating of Kv1.3 Channels of T Lymphocytes in Smith-Lemli-Opitz Syndrome Andras Balajthy 1 , Sandor Somodi 1 , Maria Peter 2 , Zoltan Petho 1 , Laszlo Vigh 2 , Gyorgy Panyi 1 , Peter Hajdu 1 . 1 Department of Biophysics and Cell biology, University of Debrecen, Debrecen, Hungary, 2 Laboratory of Molecular Stress Biology, Biological Research Centre, Szeged, Hungary. The Smith-Lemli-Opitz syndrome (SLO) is a multiple congenital anomaly, caused by a decreased or abolished activity of 7-dehydrocholesterol (7DHC) reductase, which results in the accumulation of the cholesterol precursor 7DHC in the serum and potentially in the cell membrane as well. Increased 7DHC/cholesterol ratio may modify the physico-chemical properties of plasma membrane, and hence may influence the operation of the ion channels in many cell types including T cells. To test this hypothesis we compared the biophys- ical properties of Kv1.3 channels in T cells of SLO patients (SLO-T-cells), T-cells of healthy volunteers loaded with 7DHC (7DHC-T-cells) and control T cells. The physiological consequence of altered Kv1.3 gating was measured by the proliferative capacity of CD3 þ lymphocytes. T lymphocytes were isolated from the peripheral blood of healthy volunteers (age-matched controls) and patients with SLO. 7DHC elevation in T lympho- cytes membrane was achieved upon treatment with cyclodextrin/7DHC complex. Kv1.3 currents were measured using whole-cell patch-clamp. Prolif- eration rate of lymphocytes was assessed with CFSE-dilution assay upon anti- CD3/anti-CD28 stimulation. Our results showed that both activation and inactivation kinetics were signifi- cantly slower, and the midpoint of the steady-state activation was shifted toward positive voltages in the SLO-T-cells compared to control (tau a,SLO : 0.72 ms, tau a,c : 0.60 ms ; tau i,SLO : 238,48 ms, tau i,c : 213,19 ms; V 1/2,SLO :- 21.762 mV ,V 1/2,c : - 27.98 mV). Qualitatively and quantitatively differences in the gating of Kv1.3 channels were observed in 7DHC-T-cells vs. control. T-cells from SLO patients had decreased proliferation rate as compared to healthy controls. These data demonstrate that elevated 7DHC level of cell membrane can modify the operation of ion channels and may contribute to the neurodegenerative defects in SLO. (Mecenatura OSTRAT/260/2012, TA ´ MOP 4.2.4.A/2-11-1-2012-0001). 2789-Pos Board B481 Localization of the Plasmodium Falciparum K D Channel, Pfkch1 Matias Martin 1 , Mark Luciani 1 , Anders Hay-Schmidt 2 , Peter Ellekvist 1 , Dan A. Klaerke 1 . 1 Dept. of Physiology and Biochemistry, University of Copenhagen, Frederiksberg, Denmark, 2 The Panum Institute, University of Copenhagen, Copenhagen, Denmark. The causative agent of malaria is the unicellular protozoan Plasmodium. The parasite has a complex life cycle, involving asexual replication in human red blood cells (RBCs) as well as sexual replication, forming egg-like cells called oocysts in the mosquito vector. In endemic areas it is estimated that 250 million people get infected with malaria annually, resulting in 500.000-1.000.000 deaths. Increasing resistance towards known antimalarial drugs poses a signif- icant problem in the fight against malaria. Therefore, the development of novel drugs that target vital proteins encoded by the parasite has attracted major atten- tion. Plasmodium falciparum, the species responsible for the majority of malaria- associated fatalities, encodes two putative K þ channels, PfKch1 and PfKch2, which have been cloned in our laboratories. Although viable in all intraerythrocytic stages, Kch1-null P. berghei parasites exhibit a total inhibition of oocyst development in the mosquito midgut. Thus, Kch1 might serve as a potential target in novel parasite transmission-blocking strategies. However, earlier published immunofluorescence microscopy images have suggested that Kch1 is located in the infected human red blood cell membrane. In the pre- sent study, polyclonal antibodies were raised against the PfKch1 channel and our results demonstrate that Kch1 is located in the parasites plasma membrane in all blood stages of malaria. This finding is in accordance with functional data previously published from our lab. Thus, Kch1 may be a major K þ transporter in the parasite plasma membrane and play an important role for regulation of the membrane potential of the Plasmodium parasite. 2790-Pos Board B482 Malignant Lymphoblasts in T Cell Acute Lymphoblastic Leukemia Express High Levels of Kv1.3 Eva Groessinger 1,2 , Lukas Weiss 2 , Mingyi Chen 3 , Heike Wulff 4 , Richard Greil 2 , Hubert Kerschbaum 1 . 1 Cell Biology, University of Salzburg, Salzburg, Austria, 2 3rd Medical Department, Laboratory for Immunological and Molecular Cancer Research, Paracelsus Medical University, Salzburg, Austria, 3 Department of Pathology and Laboratory Medicine, UC Davis Health System, Sacramento, CA, USA, 4 Department of Pharmacology, University of Davis, Davis, CA, USA. Acute lymploblastic leukemia (ALL) is the most common malignancy in child- hood and young adolescence, comprising about 30% of all cancers. Despite the fact that treatment strategies have strongly improved in the last decades, ALL is still one of the major causes of cancer-related death in children. Whilst the sub- types of ALL deriving from B cells are already well described by their genetic and molecular features, the biology of the T cell-linage (T-ALL) is very hetero- geneous and with the exception of some frequently occurring genetic aberra- tions until now poorly understood. By controlling the membrane potential, K þ channels regulate Ca 2þ signaling and subsequent proliferation in human lymphocytes. Expression levels of the two major K þ channels found in lymphocytes (Kv1.3 and KCa3.1) are subtype-specific. The more abundantly expressed channel typically dominates the Ca 2þ signaling events that result in proliferation. This renders the cells sensitive to specific blockade of this more highly expressed K þ channel. We investigated lymph node and bone marrow samples of T-ALL patients and found high expression levels of Kv1.3 channels in malignant T-lymphoblasts, confirmed by immunohistochemistry and immunofluorescence microscopy. Moreover, we also detected high Kv1.3 currents in a T-ALL cell line (Molt-4) compared to low KCa.3.1 currents, which was confirmed by mRNA data, as well as fluorescence microscopy. The functional role of Kv1.3 in pro- liferation of malignant T-lymphoblasts was elucidated by the pharmacological blockade of Kv1.3. Proliferation rates were significantly diminished by treat- ment with PAP-1 and ShK-L5 (specific blockers for Kv1.3 channels) but not by TRAM-34 or ICA-17043 (specific blockers for KCa3.1). Further investiga- tion might thus suggest a role for Kv1.3 channels in the treatment of T-ALL. Supported by RO1 GM076063 from the National Institute of Health and the Austrian National Bank Jubilaeumsfons No. 14311. 2791-Pos Board B483 Margatoxin is a Nonselective Inhibitor of Kv1.3 Channels - A Comprehen- sive Study Adam Bartok, Agnes Toth, Peter Hajdu, Zoltan Varga, Gyorgy Panyi. Biophysics and Cell Biology, University of Debrecen, Debrecen, Hungary. Ion channel inhibitor peptide toxins have become lead compounds for potential therapeutic use in the last decade. In the case of Kv1.3, the voltage-gated K þ Tuesday, February 18, 2014 551a
Transcript of Localization of the Plasmodium Falciparum K+ Channel, Pfkch1
Tuesday, February 18, 2014 551a
Our results point to KCa1.1 channels as a promising new target for RAtherapy and open the possibility for treatments that would not induceimmunosuppression.
2787-Pos Board B479The Functional Swtich in Potassium Channels in Myotonic DystrophyType 1 Impairs Proliferation, Migration and Fusion During MyogenesisRajeev B. Tajhya, Xueyou Hu, Mark R. Tanner, Lubov Timchenko,Christine Beeton.Baylor College of Medicine, Houston, TX, USA.Voltage-gated Kþ channels (Kv) are responsible for myoblasts proliferationand differentiation by triggering changes in membrane potential and cellvolume. Since individuals with myotonic dystrophy type 1 (DM1) displayreduced myogenesis led by prolonged myoblasts proliferation and delayedmyotubes fusion, we investigated the roles of Kþ channels in primary humanmyoblasts obtained from DM1 patients and healthy volunteers. DM1 is anautosomal dominant neuromuscular disorder affecting 1 in 8000 people world-wide. It is the most common adult-onset muscular dystrophy and currentlyhas no treatment. DM1 is characterized by muscle wasting and multi-systemdisorders.We have identified a switch in functional potassium channel expression fromKCa1.1 to Kv1 channels when comparing myoblasts from healthy individualsto myoblasts from patients with DM1. We showed increase in Kv1.2 and Kv1.5channels, and decrease in KCa1.1 channels in DM1 myoblasts at mRNA levelby RT-PCR, at protein level by immunofluorescence, and at channel activityby patch-clamp technique. We hypothesized that this switch in Kþ channelsplays a role in the reduced myogenesis observed in patients with DM1, andthat selective inhibition of Kv1 channels rescues the pathological features ofDM1 in skeletal muscle.We show that pharmacological inhibition of Kv1 channels in DM1 myoblastsnormalized proliferation, rescued matrix metalloproteinase-2 (MMP-2, aprotease necessary for myotube fusion) production, and partially rescuedmyotube fusion shown as increase in fusion index. On the contrary, selectiveinhibition of KCa1.1 in normal myoblasts lowered MMP-2 production,impaired wound healing repair, and decreased myotubes formation. Thereforewe conclude that loss of KCa1.1 and up-regulation of Kv1 channels in DM1impairs early stage of myogenesis and can be partially rescued by modulatingsuch Kþ channels.
2788-Pos Board B480Altered Gating of Kv1.3 Channels of T Lymphocytes in Smith-Lemli-OpitzSyndromeAndras Balajthy1, Sandor Somodi1, Maria Peter2, Zoltan Petho1,Laszlo Vigh2, Gyorgy Panyi1, Peter Hajdu1.1Department of Biophysics and Cell biology, University of Debrecen,Debrecen, Hungary, 2Laboratory of Molecular Stress Biology, BiologicalResearch Centre, Szeged, Hungary.The Smith-Lemli-Opitz syndrome (SLO) is a multiple congenital anomaly,caused by a decreased or abolished activity of 7-dehydrocholesterol (7DHC)reductase, which results in the accumulation of the cholesterol precursor7DHC in the serum and potentially in the cell membrane as well. Increased7DHC/cholesterol ratio may modify the physico-chemical properties of plasmamembrane, and hence may influence the operation of the ion channels in manycell types including T cells. To test this hypothesis we compared the biophys-ical properties of Kv1.3 channels in T cells of SLO patients (SLO-T-cells),T-cells of healthy volunteers loaded with 7DHC (7DHC-T-cells) and controlT cells. The physiological consequence of altered Kv1.3 gating was measuredby the proliferative capacity of CD3þ lymphocytes.T lymphocytes were isolated from the peripheral blood of healthy volunteers(age-matched controls) and patients with SLO. 7DHC elevation in T lympho-cytes membrane was achieved upon treatment with cyclodextrin/7DHCcomplex. Kv1.3 currents were measured using whole-cell patch-clamp. Prolif-eration rate of lymphocytes was assessed with CFSE-dilution assay upon anti-CD3/anti-CD28 stimulation.Our results showed that both activation and inactivation kinetics were signifi-cantly slower, and the midpoint of the steady-state activation was shiftedtoward positive voltages in the SLO-T-cells compared to control (taua,SLO:0.72 ms, taua,c: 0.60 ms ; taui,SLO: 238,48 ms, taui,c : 213,19 ms; V1/2,SLO : -21.762 mV ,V1/2,c : - 27.98 mV). Qualitatively and quantitatively differencesin the gating of Kv1.3 channels were observed in 7DHC-T-cells vs. control.T-cells from SLO patients had decreased proliferation rate as compared tohealthy controls. These data demonstrate that elevated 7DHC level of cellmembrane can modify the operation of ion channels and may contribute tothe neurodegenerative defects in SLO. (Mecenatura OSTRAT/260/2012,TAMOP 4.2.4.A/2-11-1-2012-0001).
2789-Pos Board B481Localization of the Plasmodium Falciparum KD Channel, Pfkch1Matias Martin1, Mark Luciani1, Anders Hay-Schmidt2, Peter Ellekvist1,Dan A. Klaerke1.1Dept. of Physiology and Biochemistry, University of Copenhagen,Frederiksberg, Denmark, 2The Panum Institute, University of Copenhagen,Copenhagen, Denmark.The causative agent of malaria is the unicellular protozoan Plasmodium. Theparasite has a complex life cycle, involving asexual replication in human redblood cells (RBCs) as well as sexual replication, forming egg-like cells calledoocysts in the mosquito vector. In endemic areas it is estimated that 250 millionpeople get infected with malaria annually, resulting in 500.000-1.000.000deaths. Increasing resistance towards known antimalarial drugs poses a signif-icant problem in the fight against malaria. Therefore, the development of noveldrugs that target vital proteins encoded by the parasite has attracted major atten-tion. Plasmodium falciparum, the species responsible for the majority ofmalaria- associated fatalities, encodes two putative Kþ channels, PfKch1 andPfKch2, which have been cloned in our laboratories. Although viable in allintraerythrocytic stages, Kch1-null P. berghei parasites exhibit a total inhibitionof oocyst development in the mosquito midgut. Thus, Kch1 might serve as apotential target in novel parasite transmission-blocking strategies. However,earlier published immunofluorescence microscopy images have suggestedthat Kch1 is located in the infected human red blood cell membrane. In the pre-sent study, polyclonal antibodies were raised against the PfKch1 channel andour results demonstrate that Kch1 is located in the parasites plasma membranein all blood stages of malaria. This finding is in accordance with functional datapreviously published from our lab. Thus, Kch1 may be a major Kþ transporterin the parasite plasma membrane and play an important role for regulation ofthe membrane potential of the Plasmodium parasite.
2790-Pos Board B482Malignant Lymphoblasts in T Cell Acute Lymphoblastic LeukemiaExpress High Levels of Kv1.3Eva Groessinger1,2, Lukas Weiss2, Mingyi Chen3, Heike Wulff4,Richard Greil2, Hubert Kerschbaum1.1Cell Biology, University of Salzburg, Salzburg, Austria, 23rd MedicalDepartment, Laboratory for Immunological and Molecular Cancer Research,Paracelsus Medical University, Salzburg, Austria, 3Department of Pathologyand Laboratory Medicine, UC Davis Health System, Sacramento, CA, USA,4Department of Pharmacology, University of Davis, Davis, CA, USA.Acute lymploblastic leukemia (ALL) is the most common malignancy in child-hood and young adolescence, comprising about 30% of all cancers. Despite thefact that treatment strategies have strongly improved in the last decades, ALL isstill one of the major causes of cancer-related death in children. Whilst the sub-types of ALL deriving from B cells are already well described by their geneticand molecular features, the biology of the T cell-linage (T-ALL) is very hetero-geneous and with the exception of some frequently occurring genetic aberra-tions until now poorly understood.By controlling the membrane potential, Kþ channels regulate Ca2þ signalingand subsequent proliferation in human lymphocytes. Expression levels of thetwo major Kþ channels found in lymphocytes (Kv1.3 and KCa3.1) aresubtype-specific. The more abundantly expressed channel typically dominatesthe Ca2þ signaling events that result in proliferation. This renders the cellssensitive to specific blockade of this more highly expressed Kþ channel. Weinvestigated lymph node and bone marrow samples of T-ALL patients andfound high expression levels of Kv1.3 channels in malignant T-lymphoblasts,confirmed by immunohistochemistry and immunofluorescence microscopy.Moreover, we also detected high Kv1.3 currents in a T-ALL cell line(Molt-4) compared to low KCa.3.1 currents, which was confirmed by mRNAdata, as well as fluorescence microscopy. The functional role of Kv1.3 in pro-liferation of malignant T-lymphoblasts was elucidated by the pharmacologicalblockade of Kv1.3. Proliferation rates were significantly diminished by treat-ment with PAP-1 and ShK-L5 (specific blockers for Kv1.3 channels) but notby TRAM-34 or ICA-17043 (specific blockers for KCa3.1). Further investiga-tion might thus suggest a role for Kv1.3 channels in the treatment of T-ALL.Supported by RO1 GM076063 from the National Institute of Health and theAustrian National Bank Jubilaeumsfons No. 14311.
2791-Pos Board B483Margatoxin is a Nonselective Inhibitor of Kv1.3 Channels - A Comprehen-sive StudyAdam Bartok, Agnes Toth, Peter Hajdu, Zoltan Varga, Gyorgy Panyi.Biophysics and Cell Biology, University of Debrecen, Debrecen, Hungary.Ion channel inhibitor peptide toxins have become lead compounds for potentialtherapeutic use in the last decade. In the case of Kv1.3, the voltage-gated Kþ
