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P A V Anderson - One of the best experts on this subject based on the ideXlab platform.
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Cloning and functIonal expressIon of Voltage-Gated Ion Channel subunits from cnidocytes of the Portuguese Man O'War Physalia physalis.
Journal of Experimental Biology, 2006Co-Authors: C Bouchard, R B Price, C G Moneypenny, L F Thompson, M Zillhardt, L Stalheim, P A V AndersonAbstract:SUMMARY Cnidocytes were dissociated from the tentacles of the Portuguese Man O9War Physalia physalis using heat treatment, and purified using density centrifugatIon. Visual observatIon confirmed that these cnidocytes contained a nucleus, a cnidocyst and an apical stereocilium, confirming that the cells were intact. A cnidocyte-specific amplified cDNA library was then prepared using RNA isolated from the cnidocytes, and screened for Voltage-Gated Ion Channel subunits using conventIonal molecular cloning techniques. A variety of Channel proteins were identified and full-length sequence obtained for two of them, a Ca 2+ Channel β subunit ( Pp Ca V β) and a Shaker -like K + Channel ( Pp K V 1). The locatIon of the transcripts was confirmed by RT-PCR of total RNA isolated from individually selected and rinsed cnidocytes. The functIonal properties of these two Channel proteins were characterized electrophysiologically using heterologous expressIon. Pp Ca V β modulates currents carried by both cnidarian and mammalian α 1 subunits although the specifics of the modulatIon differ. Pp K V 1 produces fast transient outward currents that have properties typical of other Shaker Channels. The possible role of these Channel proteins in the behavior of cnidocytes is discussed.
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Cloning and functIonal expressIon of Voltage-Gated Ion Channel subunits from cnidocytes of the Portuguese Man O'War Physalia physalis.
The Journal of experimental biology, 2006Co-Authors: C Bouchard, R B Price, C G Moneypenny, L F Thompson, M Zillhardt, L Stalheim, P A V AndersonAbstract:Cnidocytes were dissociated from the tentacles of the Portuguese Man O'War Physalia physalis using heat treatment, and purified using density centrifugatIon. Visual observatIon confirmed that these cnidocytes contained a nucleus, a cnidocyst and an apical stereocilium, confirming that the cells were intact. A cnidocyte-specific amplified cDNA library was then prepared using RNA isolated from the cnidocytes, and screened for Voltage-Gated Ion Channel subunits using conventIonal molecular cloning techniques. A variety of Channel proteins were identified and full-length sequence obtained for two of them, a Ca(2+) Channel beta subunit (PpCa(V)beta) and a Shaker-like K(+) Channel (PpK(V)1). The locatIon of the transcripts was confirmed by RT-PCR of total RNA isolated from individually selected and rinsed cnidocytes. The functIonal properties of these two Channel proteins were characterized electrophysiologically using heterologous expressIon. PpCa(V)beta modulates currents carried by both cnidarian and mammalian alpha(1) subunits although the specifics of the modulatIon differ. PpK(V)1 produces fast transient outward currents that have properties typical of other Shaker Channels. The possible role of these Channel proteins in the behavior of cnidocytes is discussed.
C Bouchard - One of the best experts on this subject based on the ideXlab platform.
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Cloning and functIonal expressIon of Voltage-Gated Ion Channel subunits from cnidocytes of the Portuguese Man O'War Physalia physalis.
Journal of Experimental Biology, 2006Co-Authors: C Bouchard, R B Price, C G Moneypenny, L F Thompson, M Zillhardt, L Stalheim, P A V AndersonAbstract:SUMMARY Cnidocytes were dissociated from the tentacles of the Portuguese Man O9War Physalia physalis using heat treatment, and purified using density centrifugatIon. Visual observatIon confirmed that these cnidocytes contained a nucleus, a cnidocyst and an apical stereocilium, confirming that the cells were intact. A cnidocyte-specific amplified cDNA library was then prepared using RNA isolated from the cnidocytes, and screened for Voltage-Gated Ion Channel subunits using conventIonal molecular cloning techniques. A variety of Channel proteins were identified and full-length sequence obtained for two of them, a Ca 2+ Channel β subunit ( Pp Ca V β) and a Shaker -like K + Channel ( Pp K V 1). The locatIon of the transcripts was confirmed by RT-PCR of total RNA isolated from individually selected and rinsed cnidocytes. The functIonal properties of these two Channel proteins were characterized electrophysiologically using heterologous expressIon. Pp Ca V β modulates currents carried by both cnidarian and mammalian α 1 subunits although the specifics of the modulatIon differ. Pp K V 1 produces fast transient outward currents that have properties typical of other Shaker Channels. The possible role of these Channel proteins in the behavior of cnidocytes is discussed.
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Cloning and functIonal expressIon of Voltage-Gated Ion Channel subunits from cnidocytes of the Portuguese Man O'War Physalia physalis.
The Journal of experimental biology, 2006Co-Authors: C Bouchard, R B Price, C G Moneypenny, L F Thompson, M Zillhardt, L Stalheim, P A V AndersonAbstract:Cnidocytes were dissociated from the tentacles of the Portuguese Man O'War Physalia physalis using heat treatment, and purified using density centrifugatIon. Visual observatIon confirmed that these cnidocytes contained a nucleus, a cnidocyst and an apical stereocilium, confirming that the cells were intact. A cnidocyte-specific amplified cDNA library was then prepared using RNA isolated from the cnidocytes, and screened for Voltage-Gated Ion Channel subunits using conventIonal molecular cloning techniques. A variety of Channel proteins were identified and full-length sequence obtained for two of them, a Ca(2+) Channel beta subunit (PpCa(V)beta) and a Shaker-like K(+) Channel (PpK(V)1). The locatIon of the transcripts was confirmed by RT-PCR of total RNA isolated from individually selected and rinsed cnidocytes. The functIonal properties of these two Channel proteins were characterized electrophysiologically using heterologous expressIon. PpCa(V)beta modulates currents carried by both cnidarian and mammalian alpha(1) subunits although the specifics of the modulatIon differ. PpK(V)1 produces fast transient outward currents that have properties typical of other Shaker Channels. The possible role of these Channel proteins in the behavior of cnidocytes is discussed.
Pedro Beltran-alvarez - One of the best experts on this subject based on the ideXlab platform.
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Interplay between R513 methylatIon and S516 phosphorylatIon of the cardiac Voltage-Gated sodium Channel
Amino Acids, 2014Co-Authors: Pedro Beltran-alvarez, Ferran Feixas, Sílvia Osuna, Rubí Díaz-hernández, Ramon Brugada, Sara PagansAbstract:Arginine methylatIon is a novel post-translatIonal modificatIon within the Voltage-Gated Ion Channel superfamily, including the cardiac sodium Channel, NaV1.5. We show that NaV1.5 R513 methylatIon decreases S516 phosphorylatIon rate by 4 orders of magnitude, the first evidence of protein kinase A inhibitIon by arginine methylatIon. Reciprocally, S516 phosphorylatIon blocks R513 methylatIon. NaV1.5 p.G514C, associated to cardiac conductIon disease, abrogates R513 methylatIon, while leaving S516 phosphorylatIon rate unchanged. This is the first report of methylatIon–phosphorylatIon cross-talk of a cardiac Ion Channel.
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IdentificatIon of N-terminal protein acetylatIon and arginine methylatIon of the Voltage-Gated sodium Channel in end-stage heart failure human heart.
Journal of Molecular and Cellular Cardiology, 2014Co-Authors: Pedro Beltran-alvarez, Ramon Brugada, Montserrat Batlle, Félix Pérez-villa, Anna Tarradas, Cristina Chiva, Alexandra Pérez-serra, Uwe Schulte, Eduard Sabidó, Sara PagansAbstract:The α subunit of the cardiac Voltage-Gated sodium Channel, NaV1.5, provides the rapid sodium inward current that initiates cardiomyocyte actIon potentials. Here, we analyzed for the first time the post-translatIonal modificatIons of NaV1.5 purified from end-stage heart failure human cardiac tissue. We identified R526 methylatIon as the major post-translatIonal modificatIon of any NaV1.5 arginine or lysine residue. Unexpectedly, we found that the N terminus of NaV1.5 was: 1) devoid of the initiatIon methIonine, and 2) acetylated at the resulting initial alanine residue. This is the first evidence for N-terminal acetylatIon in any member of the Voltage-Gated Ion Channel superfamily. Our results open the door to explore NaV1.5 N-terminal acetylatIon and arginine methylatIon levels as drivers or markers of end-stage heart failure.
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Protein arginine methyl transferases-3 and -5 increase cell surface expressIon of cardiac sodium Channel.
FEBS Letters, 2013Co-Authors: Pedro Beltran-alvarez, Alexsandra Espejo, Ralf Schmauder, Carlos Beltrán, Ralf Mrowka, Thomas Linke, Montserrat Batlle, Félix Pérez-villa, Guillermo J. Pérez, Fabiana S. ScornikAbstract:The α-subunit of the cardiac Voltage-Gated sodium Channel (NaV1.5) plays a central role in cardiomyocyte excitability. We have recently reported that NaV1.5 is post-translatIonally modified by arginine methylatIon. Here, we aimed to identify the enzymes that methylate NaV1.5, and to describe the role of arginine methylatIon on NaV1.5 functIon. Our results show that protein arginine methyl transferase (PRMT)-3 and -5 methylate NaV1.5 in vitro, interact with NaV1.5 in human embryonic kidney (HEK) cells, and increase NaV1.5 current density by enhancing NaV1.5 cell surface expressIon. Our observatIons are the first evidence of regulatIon of a Voltage-Gated Ion Channel, including calcium, potassium, sodium and TRP Channels, by arginine methylatIon.
Daniel L Minor - One of the best experts on this subject based on the ideXlab platform.
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k2p2 1 trek 1 activator complexes reveal a cryptic selectivity filter binding site
Nature, 2017Co-Authors: Marco Lolicato, Cristina Arrigoni, Takahiro Mori, Yoko Sekioka, Clifford Bryant, Kimberly A Clark, Daniel L MinorAbstract:Crystal structures of an activated two-pore potassium Channel reveal a cryptic binding pocket that binds small-molecule activators that restrict the mobility of the selectivity filter and surrounding structure, stabilizing an active ‘leak-mode’ conformatIon. The two-pore domain potassium Channels (K2P) are dimeric Channels of the Voltage-Gated Ion Channel superfamily and are responsive to temperature, mechanical stimuli and small molecules. Despite their physiological importance in pain and temperature perceptIon as well as mood regulatIon, their pharmacology (how small molecules modulate their activity) is not well understood. In this work, crystal structures of an activated K2P Channel are reported, revealing a cryptic binding pocket behind the selectivity filter of the chanel. The small-molecule activators bind to this pocket and restrict the mobility of the selectivity filter and the surrounding structure. This stabilizes a 'leak-mode' conformatIon, activating the Channel and allowing current to leak through. Polymodal thermo- and mechanosensitive two-pore domain potassium (K2P) Channels of the TREK1 subfamily generate ‘leak’ currents that regulate neuronal excitability, respond to lipids, temperature and mechanical stretch, and influence pain, temperature perceptIon and anaesthetic responses1,2,3. These dimeric Voltage-Gated Ion Channel (VGIC) superfamily members have a unique topology comprising two pore-forming regIons per subunit4,5,6. In contrast to other potassium Channels, K2P Channels use a selectivity filter ‘C-type’ gate7,8,9,10 as the principal gating site. Despite recent advances3,11,12, poor pharmacological profiles of K2P Channels limit mechanistic and biological studies. Here we describe a class of small-molecule TREK activators that directly stimulate the C-type gate by acting as molecular wedges that restrict interdomain interface movement behind the selectivity filter. Structures of K2P2.1 (also known as TREK-1) alone and with two selective K2P2.1 (TREK-1) and K2P10.1 (TREK-2) activators—an N-aryl-sulfonamide, ML335, and a thiophene-carboxamide, ML402—define a cryptic binding pocket unlike other Ion Channel small-molecule binding sites and, together with functIonal studies, identify a catIon–π interactIon that controls selectivity. Together, our data reveal a druggable K2P site that stabilizes the C-type gate ‘leak mode’ and provide direct evidence for K2P selectivity filter gating.
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k2p2 1 trek 1 activator complexes reveal a cryptic selectivity filter binding site
Nature, 2017Co-Authors: Marco Lolicato, Cristina Arrigoni, Takahiro Mori, Yoko Sekioka, Clifford Bryant, Kimberly A Clark, Daniel L MinorAbstract:Polymodal thermo- and mechanosensitive two-pore domain potassium (K2P) Channels of the TREK subfamily generate 'leak' currents that regulate neuronal excitability, respond to lipids, temperature and mechanical stretch, and influence pain, temperature perceptIon and anaesthetic responses. These dimeric Voltage-Gated Ion Channel (VGIC) superfamily members have a unique topology comprising two pore-forming regIons per subunit. In contrast to other potassium Channels, K2P Channels use a selectivity filter 'C-type' gate as the principal gating site. Despite recent advances, poor pharmacological profiles of K2P Channels limit mechanistic and biological studies. Here we describe a class of small-molecule TREK activators that directly stimulate the C-type gate by acting as molecular wedges that restrict interdomain interface movement behind the selectivity filter. Structures of K2P2.1 (also known as TREK-1) alone and with two selective K2P2.1 (TREK-1) and K2P10.1 (TREK-2) activators-an N-aryl-sulfonamide, ML335, and a thiophene-carboxamide, ML402-define a cryptic binding pocket unlike other Ion Channel small-molecule binding sites and, together with functIonal studies, identify a catIon-π interactIon that controls selectivity. Together, our data reveal a druggable K2P site that stabilizes the C-type gate 'leak mode' and provide direct evidence for K2P selectivity filter gating.
David J Schulz - One of the best experts on this subject based on the ideXlab platform.
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effects of chronic spinal cord injury on relatIonships among Ion Channel and receptor mrnas in mouse lumbar spinal cord
Neuroscience, 2018Co-Authors: Virginia B Garcia, Matthew D Abbinanti, Ronald M Harriswarrick, David J SchulzAbstract:Spinal cord injury (SCI) causes widespread changes in gene expressIon of the spinal cord, even in the undamaged spinal cord below the level of the lesIon. Less is known about changes in the correlated expressIon of genes after SCI. We investigated gene co-expressIon networks among Voltage-Gated Ion Channel and neurotransmitter receptor mRNA levels using quantitative RT-PCR in longitudinal slices of the mouse lumbar spinal cord in control and chronic SCI animals. These longitudinal slices were made from the ventral surface of the cord, thus forming slices relatively enriched in motor neurons or interneurons. We performed absolute quantitatIon of mRNA copy number for 50 Ion Channel or receptor transcripts from each sample, and used multiple correlatIon analyses to detect patterns in correlated mRNA levels across all pairs of genes. The majority of Channels and receptors changed in expressIon as a result of chronic SCI, but did so differently across slice levels. Furthermore, motor neuron-enriched slices experienced an overall loss of correlated Channel and receptor expressIon, while interneuron slices showed a dramatic increase in the number of positively correlated transcripts. These correlatIon profiles suggest that spinal cord injury induces distinct changes across cell types in the organizatIon of gene co-expressIon networks for Ion Channels and transmitter receptors.
