The Experts below are selected from a list of 213 Experts worldwide ranked by ideXlab platform
Ana M. Correa - One of the best experts on this subject based on the ideXlab platform.
-
Is the Skeletal Muscle Sodium Channel of the Batrachotoxin (BTX)-Producing Phyllobates aurotaenia Poison Dart Frog Resistant to BTX?
Biophysical Journal, 2010Co-Authors: Ludivine Frezza, Leonardo Fierro, Santiago Castaño, Francisco Bezanilla, Helberg Asencio, Walter Sandtner, Ana M. CorreaAbstract:Batrachotoxin is a potent toxin found in skins of Phyllobates frogs. The skeletal muscle Na+ channels of Phyllobates aurotaenia frogs have been proposed to be resistant to high concentrations of BTX (>1μM). In order to unravel the mechanism and structural elements that confer BTX-resistance to P. aurotaenia, we cloned its skeletal muscle Na+ channel, PaNaV1.4. As reported last year, PaNaV1.4 has high homology (>70%) with other NaV1.4 channels especially in the membrane spanning regions. Some residues that have been identified in mutagenesis studies as critical for BTX-channel interaction in mammalian NaV are conserved in PaNaV1.4. To further address the issue, we have expressed PaNaV1.4 in Xenopus laevis oocytes. We report here the functional characterization of PaNaV1.4, studied under voltage-clamp, and its response to BTX.PaNaV1.4 expresses robustly. While the general characteristics of the ionic currents were similar, at room temperature PaNaV1.4 tended to open at more depolarized voltages, inactivated faster and currents peaked earlier than rNaV1.4. BTX, at concentrations as high as 10μM, had a significantly lower effect on PaNaV1.4 currents than on rNaV1.4. The ratio of plateau to peak currents at 80 mV was ∼0.2-0.5 in PaNaV1.4 while >0.95 in rNaV1.4. BTX modification of PaNaV1.4 occurred at a slow rate. Both activation thresholds were negatively shifted. Because most of the residues proposed to participate in the BTX effect are located in the pore lining segment (S6) of NaV, we have also studied Pa/rNaV1.4 hybrid channels with domains, S6 segments or residues swapped or exchanged.Supported by COLCIENCIAS1106-12-13836 (LF), AHA 0725763Z (WS), and NIH GM030376 (FB) and GM068044 (AMC).
-
cloning and sequence analysis of the voltage gated muscle na channel from the poison dart frog Phyllobates aurotaenia
Biophysical Journal, 2009Co-Authors: Leonardo Fierro, Santiago Castaño, Ludivine Frezza, Alain J. Labro, Francisco Bezanilla, Ana M. CorreaAbstract:Poison dart frogs of the genus Phyllobates secrete lipophilic alkaloid toxins through their skin that were used by Colombian Amerindians to poison the tips of blowdarts. One of the most potent toxins identified is batrachotoxin (BTX) which is an activator of voltage-gated Na+ channels. BTX causes sustained opening of these channels by shifting the voltage-dependent activation to more hyperpolarized potentials and by disabling both fast and slow inactivation. It also alters pore conductance and selectivity. Endogenous Na+ channels of the poison arrow frog have been proposed to be insensitive to lethal amounts of BTX. In this project we aim to identify what confers BTX insensitivity to Na+ channels of the host frog Phyllobates aurotaenia, therefore we cloned its skeletal muscle NaV channel. Total RNA from skeletal muscle of Phyllobates aurotaenia was isolated and cDNA was obtained with degenerate primers.The 1819 amino acids sequence shares 72% sequence identity with the rat Na+ channel NaV1.4, and 73% with that of the snake Thamnophis sirtalis. The TMs are extremely well conserved (87%) with absolute conservation of S4 in all domains. The N-and C-termini as well as the cytoplasmic linkers between domains are more divergent. The D3-D4 linker containing the IFM motif is highly conserved except for Q1348E and K1350P. The DEKA-motif is also absolutely conserved as are the GGGS gating hinge and the QGFS motifs. BTX is thought to bind in the pore region, from the selectivity filter ring to the pore lining S6 TMs. We have identified two S to A mutations flanking the gating-hinge in domains 1 and 3 that may participate in toxin-insensitivity of the Phyllobates channel by impairing the binding of BTX. Supported by NIH GM68044(AMC) and GM30376(FB) and by COLCIENCIAS1106-12-13836(LF).
-
Cloning and Sequence Analysis of the Voltage-Gated Muscle Na+ Channel from the Poison Dart Frog Phyllobates aurotaenia
Biophysical Journal, 2009Co-Authors: Santiago Castaño, Leonardo Fierro, Ludivine Frezza, Alain J. Labro, Francisco Bezanilla, Ana M. CorreaAbstract:Poison dart frogs of the genus Phyllobates secrete lipophilic alkaloid toxins through their skin that were used by Colombian Amerindians to poison the tips of blowdarts. One of the most potent toxins identified is batrachotoxin (BTX) which is an activator of voltage-gated Na+ channels. BTX causes sustained opening of these channels by shifting the voltage-dependent activation to more hyperpolarized potentials and by disabling both fast and slow inactivation. It also alters pore conductance and selectivity. Endogenous Na+ channels of the poison arrow frog have been proposed to be insensitive to lethal amounts of BTX. In this project we aim to identify what confers BTX insensitivity to Na+ channels of the host frog Phyllobates aurotaenia, therefore we cloned its skeletal muscle NaV channel. Total RNA from skeletal muscle of Phyllobates aurotaenia was isolated and cDNA was obtained with degenerate primers.The 1819 amino acids sequence shares 72% sequence identity with the rat Na+ channel NaV1.4, and 73% with that of the snake Thamnophis sirtalis. The TMs are extremely well conserved (87%) with absolute conservation of S4 in all domains. The N-and C-termini as well as the cytoplasmic linkers between domains are more divergent. The D3-D4 linker containing the IFM motif is highly conserved except for Q1348E and K1350P. The DEKA-motif is also absolutely conserved as are the GGGS gating hinge and the QGFS motifs. BTX is thought to bind in the pore region, from the selectivity filter ring to the pore lining S6 TMs. We have identified two S to A mutations flanking the gating-hinge in domains 1 and 3 that may participate in toxin-insensitivity of the Phyllobates channel by impairing the binding of BTX. Supported by NIH GM68044(AMC) and GM30376(FB) and by COLCIENCIAS1106-12-13836(LF).
Santiago Castaño - One of the best experts on this subject based on the ideXlab platform.
-
Is the Skeletal Muscle Sodium Channel of the Batrachotoxin (BTX)-Producing Phyllobates aurotaenia Poison Dart Frog Resistant to BTX?
Biophysical Journal, 2010Co-Authors: Ludivine Frezza, Leonardo Fierro, Santiago Castaño, Francisco Bezanilla, Helberg Asencio, Walter Sandtner, Ana M. CorreaAbstract:Batrachotoxin is a potent toxin found in skins of Phyllobates frogs. The skeletal muscle Na+ channels of Phyllobates aurotaenia frogs have been proposed to be resistant to high concentrations of BTX (>1μM). In order to unravel the mechanism and structural elements that confer BTX-resistance to P. aurotaenia, we cloned its skeletal muscle Na+ channel, PaNaV1.4. As reported last year, PaNaV1.4 has high homology (>70%) with other NaV1.4 channels especially in the membrane spanning regions. Some residues that have been identified in mutagenesis studies as critical for BTX-channel interaction in mammalian NaV are conserved in PaNaV1.4. To further address the issue, we have expressed PaNaV1.4 in Xenopus laevis oocytes. We report here the functional characterization of PaNaV1.4, studied under voltage-clamp, and its response to BTX.PaNaV1.4 expresses robustly. While the general characteristics of the ionic currents were similar, at room temperature PaNaV1.4 tended to open at more depolarized voltages, inactivated faster and currents peaked earlier than rNaV1.4. BTX, at concentrations as high as 10μM, had a significantly lower effect on PaNaV1.4 currents than on rNaV1.4. The ratio of plateau to peak currents at 80 mV was ∼0.2-0.5 in PaNaV1.4 while >0.95 in rNaV1.4. BTX modification of PaNaV1.4 occurred at a slow rate. Both activation thresholds were negatively shifted. Because most of the residues proposed to participate in the BTX effect are located in the pore lining segment (S6) of NaV, we have also studied Pa/rNaV1.4 hybrid channels with domains, S6 segments or residues swapped or exchanged.Supported by COLCIENCIAS1106-12-13836 (LF), AHA 0725763Z (WS), and NIH GM030376 (FB) and GM068044 (AMC).
-
cloning and sequence analysis of the voltage gated muscle na channel from the poison dart frog Phyllobates aurotaenia
Biophysical Journal, 2009Co-Authors: Leonardo Fierro, Santiago Castaño, Ludivine Frezza, Alain J. Labro, Francisco Bezanilla, Ana M. CorreaAbstract:Poison dart frogs of the genus Phyllobates secrete lipophilic alkaloid toxins through their skin that were used by Colombian Amerindians to poison the tips of blowdarts. One of the most potent toxins identified is batrachotoxin (BTX) which is an activator of voltage-gated Na+ channels. BTX causes sustained opening of these channels by shifting the voltage-dependent activation to more hyperpolarized potentials and by disabling both fast and slow inactivation. It also alters pore conductance and selectivity. Endogenous Na+ channels of the poison arrow frog have been proposed to be insensitive to lethal amounts of BTX. In this project we aim to identify what confers BTX insensitivity to Na+ channels of the host frog Phyllobates aurotaenia, therefore we cloned its skeletal muscle NaV channel. Total RNA from skeletal muscle of Phyllobates aurotaenia was isolated and cDNA was obtained with degenerate primers.The 1819 amino acids sequence shares 72% sequence identity with the rat Na+ channel NaV1.4, and 73% with that of the snake Thamnophis sirtalis. The TMs are extremely well conserved (87%) with absolute conservation of S4 in all domains. The N-and C-termini as well as the cytoplasmic linkers between domains are more divergent. The D3-D4 linker containing the IFM motif is highly conserved except for Q1348E and K1350P. The DEKA-motif is also absolutely conserved as are the GGGS gating hinge and the QGFS motifs. BTX is thought to bind in the pore region, from the selectivity filter ring to the pore lining S6 TMs. We have identified two S to A mutations flanking the gating-hinge in domains 1 and 3 that may participate in toxin-insensitivity of the Phyllobates channel by impairing the binding of BTX. Supported by NIH GM68044(AMC) and GM30376(FB) and by COLCIENCIAS1106-12-13836(LF).
-
Cloning and Sequence Analysis of the Voltage-Gated Muscle Na+ Channel from the Poison Dart Frog Phyllobates aurotaenia
Biophysical Journal, 2009Co-Authors: Santiago Castaño, Leonardo Fierro, Ludivine Frezza, Alain J. Labro, Francisco Bezanilla, Ana M. CorreaAbstract:Poison dart frogs of the genus Phyllobates secrete lipophilic alkaloid toxins through their skin that were used by Colombian Amerindians to poison the tips of blowdarts. One of the most potent toxins identified is batrachotoxin (BTX) which is an activator of voltage-gated Na+ channels. BTX causes sustained opening of these channels by shifting the voltage-dependent activation to more hyperpolarized potentials and by disabling both fast and slow inactivation. It also alters pore conductance and selectivity. Endogenous Na+ channels of the poison arrow frog have been proposed to be insensitive to lethal amounts of BTX. In this project we aim to identify what confers BTX insensitivity to Na+ channels of the host frog Phyllobates aurotaenia, therefore we cloned its skeletal muscle NaV channel. Total RNA from skeletal muscle of Phyllobates aurotaenia was isolated and cDNA was obtained with degenerate primers.The 1819 amino acids sequence shares 72% sequence identity with the rat Na+ channel NaV1.4, and 73% with that of the snake Thamnophis sirtalis. The TMs are extremely well conserved (87%) with absolute conservation of S4 in all domains. The N-and C-termini as well as the cytoplasmic linkers between domains are more divergent. The D3-D4 linker containing the IFM motif is highly conserved except for Q1348E and K1350P. The DEKA-motif is also absolutely conserved as are the GGGS gating hinge and the QGFS motifs. BTX is thought to bind in the pore region, from the selectivity filter ring to the pore lining S6 TMs. We have identified two S to A mutations flanking the gating-hinge in domains 1 and 3 that may participate in toxin-insensitivity of the Phyllobates channel by impairing the binding of BTX. Supported by NIH GM68044(AMC) and GM30376(FB) and by COLCIENCIAS1106-12-13836(LF).
Leonardo Fierro - One of the best experts on this subject based on the ideXlab platform.
-
histological description of the skin glands of Phyllobates bicolor anura dendrobatidae using three staining techniques
International Journal of Morphology, 2014Co-Authors: Freddy Morenogomez, Tania Duque, Leonardo Fierro, Juan Arango, Xiomara Peckham, Helberg AsenciosantofimioAbstract:A la fecha no existe mayor informacion con respecto a los mecanismos para obtener y almacenar las toxinas cutaneas de ranas de la familia Dendrobatidae. Con el fin de contribuir y entender como son almacenadas estas toxinas, realizamos una descripcion histologica de las glandulas cutaneas de la especie Phyllobates bicolor. La piel de dos ranas adultas se examino mediante tres tecnicas de tincion histologica (hematoxilina-eosina, PAS y tricromico de Masson) mediante microscopia optica convencional. La piel de P. bicolor contiene dos tipos de glandulas exocrinas: mucosas y serosas, que vierten sus productos a la superficie epidermica a traves de un conducto intra-epitelial que conduce a un estoma. Las glandulas mucosas, serosas y los conductos intercalados estan rodeados por una funda discontinua de celulas mioepiteliales, las que colapsan el lumen de los acinos y conductos, facilitando la secrecion y liberacion de su contenido. Las glandulas serosas tienen un sincitio polarizado de celulas epiteliales columnares cubicas. Ambas glandulas tienen una secrecion mixta, por lo tanto, los contenidos de las glandulas mucosas tienden a ser neutral y basofilas, mientras que los contenidos de las glandulas serosas son basofilas y acidofilas.
-
Histological Description of the Skin Glands of Phyllobates bicolor (Anura: Dendrobatidae) Using Three Staining Techniques Descripción Histológica de las Glándulas Cutáneas de Phyllobates bicolor (Anura: Dendrobatidae) Usando Tres Técnicas de Tinción
2014Co-Authors: Freddy Moreno-gómez, Tania Duque, Leonardo Fierro, Juan Arango, Xiomara Peckham, Helberg Asencio-santofimioAbstract:MORENO-GOMEZ, F.; DUQUE, T.; FIERRO, L.; ARANGO, J.; PECKHAM, X. & ASENCIO-SANTOFIMIO, H. Histologicaldescription of the skin glands of Phyllobates bicolor (Anura: Dendrobatidae) using three staining techniques. Int. J. Morphol., 32(3) :882-888, 2014.SUMMARY: The mechanisms to obtain and store skin toxins in frogs in of the family Dendrobatidae are not completely understood.In order to contribute to understand how toxins are stored, we provide a histological description of the cutaneous glands of the speciesPhyllobates bicolor. The skin of two adult frogs was examined through three histological staining techniques (hematoxilin-eosin, PASand Masson Trichrome) using conventional optic microscopy. The skin of Phyllobates bicolor contains two types of exocrine glands:mucous and serous, which empty their products to the epidermal surface through an intra-epithelial duct that leads to a stoma. Themucous and serous glands and the intercalated ducts are surrounded by a discontinuous sheath of myoepithelial cells, which cola pse thelumen of the acinus and the lumen of ducts and facilitate the secretion and release of their content. The serous glands have a polarizedsyncytium of tall cuboidal or columnar epithelial cells. Both glands have a mixed secretion, thus, the contents of mucous gland s tend tobe neutral and basophilic, while the contents of the serous glands are basophilic and acidophilic.KEY WORDS: Amphibians; Phyllobates bicolor ; Integument; Epidermis; Dermis; Mucous glands; Serous glands; Chemicaldefenses; Bioaccumulation.
-
Is the Skeletal Muscle Sodium Channel of the Batrachotoxin (BTX)-Producing Phyllobates aurotaenia Poison Dart Frog Resistant to BTX?
Biophysical Journal, 2010Co-Authors: Ludivine Frezza, Leonardo Fierro, Santiago Castaño, Francisco Bezanilla, Helberg Asencio, Walter Sandtner, Ana M. CorreaAbstract:Batrachotoxin is a potent toxin found in skins of Phyllobates frogs. The skeletal muscle Na+ channels of Phyllobates aurotaenia frogs have been proposed to be resistant to high concentrations of BTX (>1μM). In order to unravel the mechanism and structural elements that confer BTX-resistance to P. aurotaenia, we cloned its skeletal muscle Na+ channel, PaNaV1.4. As reported last year, PaNaV1.4 has high homology (>70%) with other NaV1.4 channels especially in the membrane spanning regions. Some residues that have been identified in mutagenesis studies as critical for BTX-channel interaction in mammalian NaV are conserved in PaNaV1.4. To further address the issue, we have expressed PaNaV1.4 in Xenopus laevis oocytes. We report here the functional characterization of PaNaV1.4, studied under voltage-clamp, and its response to BTX.PaNaV1.4 expresses robustly. While the general characteristics of the ionic currents were similar, at room temperature PaNaV1.4 tended to open at more depolarized voltages, inactivated faster and currents peaked earlier than rNaV1.4. BTX, at concentrations as high as 10μM, had a significantly lower effect on PaNaV1.4 currents than on rNaV1.4. The ratio of plateau to peak currents at 80 mV was ∼0.2-0.5 in PaNaV1.4 while >0.95 in rNaV1.4. BTX modification of PaNaV1.4 occurred at a slow rate. Both activation thresholds were negatively shifted. Because most of the residues proposed to participate in the BTX effect are located in the pore lining segment (S6) of NaV, we have also studied Pa/rNaV1.4 hybrid channels with domains, S6 segments or residues swapped or exchanged.Supported by COLCIENCIAS1106-12-13836 (LF), AHA 0725763Z (WS), and NIH GM030376 (FB) and GM068044 (AMC).
-
cloning and sequence analysis of the voltage gated muscle na channel from the poison dart frog Phyllobates aurotaenia
Biophysical Journal, 2009Co-Authors: Leonardo Fierro, Santiago Castaño, Ludivine Frezza, Alain J. Labro, Francisco Bezanilla, Ana M. CorreaAbstract:Poison dart frogs of the genus Phyllobates secrete lipophilic alkaloid toxins through their skin that were used by Colombian Amerindians to poison the tips of blowdarts. One of the most potent toxins identified is batrachotoxin (BTX) which is an activator of voltage-gated Na+ channels. BTX causes sustained opening of these channels by shifting the voltage-dependent activation to more hyperpolarized potentials and by disabling both fast and slow inactivation. It also alters pore conductance and selectivity. Endogenous Na+ channels of the poison arrow frog have been proposed to be insensitive to lethal amounts of BTX. In this project we aim to identify what confers BTX insensitivity to Na+ channels of the host frog Phyllobates aurotaenia, therefore we cloned its skeletal muscle NaV channel. Total RNA from skeletal muscle of Phyllobates aurotaenia was isolated and cDNA was obtained with degenerate primers.The 1819 amino acids sequence shares 72% sequence identity with the rat Na+ channel NaV1.4, and 73% with that of the snake Thamnophis sirtalis. The TMs are extremely well conserved (87%) with absolute conservation of S4 in all domains. The N-and C-termini as well as the cytoplasmic linkers between domains are more divergent. The D3-D4 linker containing the IFM motif is highly conserved except for Q1348E and K1350P. The DEKA-motif is also absolutely conserved as are the GGGS gating hinge and the QGFS motifs. BTX is thought to bind in the pore region, from the selectivity filter ring to the pore lining S6 TMs. We have identified two S to A mutations flanking the gating-hinge in domains 1 and 3 that may participate in toxin-insensitivity of the Phyllobates channel by impairing the binding of BTX. Supported by NIH GM68044(AMC) and GM30376(FB) and by COLCIENCIAS1106-12-13836(LF).
-
Cloning and Sequence Analysis of the Voltage-Gated Muscle Na+ Channel from the Poison Dart Frog Phyllobates aurotaenia
Biophysical Journal, 2009Co-Authors: Santiago Castaño, Leonardo Fierro, Ludivine Frezza, Alain J. Labro, Francisco Bezanilla, Ana M. CorreaAbstract:Poison dart frogs of the genus Phyllobates secrete lipophilic alkaloid toxins through their skin that were used by Colombian Amerindians to poison the tips of blowdarts. One of the most potent toxins identified is batrachotoxin (BTX) which is an activator of voltage-gated Na+ channels. BTX causes sustained opening of these channels by shifting the voltage-dependent activation to more hyperpolarized potentials and by disabling both fast and slow inactivation. It also alters pore conductance and selectivity. Endogenous Na+ channels of the poison arrow frog have been proposed to be insensitive to lethal amounts of BTX. In this project we aim to identify what confers BTX insensitivity to Na+ channels of the host frog Phyllobates aurotaenia, therefore we cloned its skeletal muscle NaV channel. Total RNA from skeletal muscle of Phyllobates aurotaenia was isolated and cDNA was obtained with degenerate primers.The 1819 amino acids sequence shares 72% sequence identity with the rat Na+ channel NaV1.4, and 73% with that of the snake Thamnophis sirtalis. The TMs are extremely well conserved (87%) with absolute conservation of S4 in all domains. The N-and C-termini as well as the cytoplasmic linkers between domains are more divergent. The D3-D4 linker containing the IFM motif is highly conserved except for Q1348E and K1350P. The DEKA-motif is also absolutely conserved as are the GGGS gating hinge and the QGFS motifs. BTX is thought to bind in the pore region, from the selectivity filter ring to the pore lining S6 TMs. We have identified two S to A mutations flanking the gating-hinge in domains 1 and 3 that may participate in toxin-insensitivity of the Phyllobates channel by impairing the binding of BTX. Supported by NIH GM68044(AMC) and GM30376(FB) and by COLCIENCIAS1106-12-13836(LF).
Ging Kuo Wang - One of the best experts on this subject based on the ideXlab platform.
-
single rat muscle na channel mutation confers batrachotoxin autoresistance found in poison dart frog Phyllobates terribilis
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Sho-ya Wang, Ging Kuo WangAbstract:Poison-dart Phyllobates terribilis frogs sequester lethal amounts of steroidal alkaloid batrachotoxin (BTX) in their skin as a defense mechanism against predators. BTX targets voltage-gated Na+ channels and enables them to open persistently. How BTX autoresistance arises in such frogs remains a mystery. The BTX receptor has been delineated along the Na+ channel inner cavity, which is formed jointly by four S6 transmembrane segments from domains D1 to D4. Within the P. terribilis muscle Na+ channel, five amino acid (AA) substitutions have been identified at D1/S6 and D4/S6. We therefore investigated the role of these naturally occurring substitutions in BTX autoresistance by introducing them into rat Nav1.4 muscle Na+ channel, both individually and in combination. Our results showed that combination mutants containing an N1584T substitution all conferred a complete BTX-resistant phenotype when expressed in mammalian HEK293t cells. The single N1584T mutant also retained its functional integrity and became exceptionally resistant to 5 µM BTX, aside from a small residual BTX effect. Single and combination mutants with the other four S6 residues (S429A, I433V, A445D, and V1583I) all remained highly BTX sensitive. These findings, along with diverse BTX phenotypes of N1584K/A/D/T mutant channels, led us to conclude that the conserved N1584 residue is indispensable for BTX actions, probably functioning as an integral part of the BTX receptor. Thus, complete BTX autoresistance found in P. terribilis muscle Na+ channels could emerge primarily from a single AA substitution (asparagine→threonine) via a single nucleotide mutation (AAC→ACC).
-
Single rat muscle Na+ channel mutation confers batrachotoxin autoresistance found in poison-dart frog Phyllobates terribilis.
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Sho-ya Wang, Ging Kuo WangAbstract:Poison-dart Phyllobates terribilis frogs sequester lethal amounts of steroidal alkaloid batrachotoxin (BTX) in their skin as a defense mechanism against predators. BTX targets voltage-gated Na+ channels and enables them to open persistently. How BTX autoresistance arises in such frogs remains a mystery. The BTX receptor has been delineated along the Na+ channel inner cavity, which is formed jointly by four S6 transmembrane segments from domains D1 to D4. Within the P. terribilis muscle Na+ channel, five amino acid (AA) substitutions have been identified at D1/S6 and D4/S6. We therefore investigated the role of these naturally occurring substitutions in BTX autoresistance by introducing them into rat Nav1.4 muscle Na+ channel, both individually and in combination. Our results showed that combination mutants containing an N1584T substitution all conferred a complete BTX-resistant phenotype when expressed in mammalian HEK293t cells. The single N1584T mutant also retained its functional integrity and became exceptionally resistant to 5 µM BTX, aside from a small residual BTX effect. Single and combination mutants with the other four S6 residues (S429A, I433V, A445D, and V1583I) all remained highly BTX sensitive. These findings, along with diverse BTX phenotypes of N1584K/A/D/T mutant channels, led us to conclude that the conserved N1584 residue is indispensable for BTX actions, probably functioning as an integral part of the BTX receptor. Thus, complete BTX autoresistance found in P. terribilis muscle Na+ channels could emerge primarily from a single AA substitution (asparagine→threonine) via a single nucleotide mutation (AAC→ACC).
Ludivine Frezza - One of the best experts on this subject based on the ideXlab platform.
-
Is the Skeletal Muscle Sodium Channel of the Batrachotoxin (BTX)-Producing Phyllobates aurotaenia Poison Dart Frog Resistant to BTX?
Biophysical Journal, 2010Co-Authors: Ludivine Frezza, Leonardo Fierro, Santiago Castaño, Francisco Bezanilla, Helberg Asencio, Walter Sandtner, Ana M. CorreaAbstract:Batrachotoxin is a potent toxin found in skins of Phyllobates frogs. The skeletal muscle Na+ channels of Phyllobates aurotaenia frogs have been proposed to be resistant to high concentrations of BTX (>1μM). In order to unravel the mechanism and structural elements that confer BTX-resistance to P. aurotaenia, we cloned its skeletal muscle Na+ channel, PaNaV1.4. As reported last year, PaNaV1.4 has high homology (>70%) with other NaV1.4 channels especially in the membrane spanning regions. Some residues that have been identified in mutagenesis studies as critical for BTX-channel interaction in mammalian NaV are conserved in PaNaV1.4. To further address the issue, we have expressed PaNaV1.4 in Xenopus laevis oocytes. We report here the functional characterization of PaNaV1.4, studied under voltage-clamp, and its response to BTX.PaNaV1.4 expresses robustly. While the general characteristics of the ionic currents were similar, at room temperature PaNaV1.4 tended to open at more depolarized voltages, inactivated faster and currents peaked earlier than rNaV1.4. BTX, at concentrations as high as 10μM, had a significantly lower effect on PaNaV1.4 currents than on rNaV1.4. The ratio of plateau to peak currents at 80 mV was ∼0.2-0.5 in PaNaV1.4 while >0.95 in rNaV1.4. BTX modification of PaNaV1.4 occurred at a slow rate. Both activation thresholds were negatively shifted. Because most of the residues proposed to participate in the BTX effect are located in the pore lining segment (S6) of NaV, we have also studied Pa/rNaV1.4 hybrid channels with domains, S6 segments or residues swapped or exchanged.Supported by COLCIENCIAS1106-12-13836 (LF), AHA 0725763Z (WS), and NIH GM030376 (FB) and GM068044 (AMC).
-
cloning and sequence analysis of the voltage gated muscle na channel from the poison dart frog Phyllobates aurotaenia
Biophysical Journal, 2009Co-Authors: Leonardo Fierro, Santiago Castaño, Ludivine Frezza, Alain J. Labro, Francisco Bezanilla, Ana M. CorreaAbstract:Poison dart frogs of the genus Phyllobates secrete lipophilic alkaloid toxins through their skin that were used by Colombian Amerindians to poison the tips of blowdarts. One of the most potent toxins identified is batrachotoxin (BTX) which is an activator of voltage-gated Na+ channels. BTX causes sustained opening of these channels by shifting the voltage-dependent activation to more hyperpolarized potentials and by disabling both fast and slow inactivation. It also alters pore conductance and selectivity. Endogenous Na+ channels of the poison arrow frog have been proposed to be insensitive to lethal amounts of BTX. In this project we aim to identify what confers BTX insensitivity to Na+ channels of the host frog Phyllobates aurotaenia, therefore we cloned its skeletal muscle NaV channel. Total RNA from skeletal muscle of Phyllobates aurotaenia was isolated and cDNA was obtained with degenerate primers.The 1819 amino acids sequence shares 72% sequence identity with the rat Na+ channel NaV1.4, and 73% with that of the snake Thamnophis sirtalis. The TMs are extremely well conserved (87%) with absolute conservation of S4 in all domains. The N-and C-termini as well as the cytoplasmic linkers between domains are more divergent. The D3-D4 linker containing the IFM motif is highly conserved except for Q1348E and K1350P. The DEKA-motif is also absolutely conserved as are the GGGS gating hinge and the QGFS motifs. BTX is thought to bind in the pore region, from the selectivity filter ring to the pore lining S6 TMs. We have identified two S to A mutations flanking the gating-hinge in domains 1 and 3 that may participate in toxin-insensitivity of the Phyllobates channel by impairing the binding of BTX. Supported by NIH GM68044(AMC) and GM30376(FB) and by COLCIENCIAS1106-12-13836(LF).
-
Cloning and Sequence Analysis of the Voltage-Gated Muscle Na+ Channel from the Poison Dart Frog Phyllobates aurotaenia
Biophysical Journal, 2009Co-Authors: Santiago Castaño, Leonardo Fierro, Ludivine Frezza, Alain J. Labro, Francisco Bezanilla, Ana M. CorreaAbstract:Poison dart frogs of the genus Phyllobates secrete lipophilic alkaloid toxins through their skin that were used by Colombian Amerindians to poison the tips of blowdarts. One of the most potent toxins identified is batrachotoxin (BTX) which is an activator of voltage-gated Na+ channels. BTX causes sustained opening of these channels by shifting the voltage-dependent activation to more hyperpolarized potentials and by disabling both fast and slow inactivation. It also alters pore conductance and selectivity. Endogenous Na+ channels of the poison arrow frog have been proposed to be insensitive to lethal amounts of BTX. In this project we aim to identify what confers BTX insensitivity to Na+ channels of the host frog Phyllobates aurotaenia, therefore we cloned its skeletal muscle NaV channel. Total RNA from skeletal muscle of Phyllobates aurotaenia was isolated and cDNA was obtained with degenerate primers.The 1819 amino acids sequence shares 72% sequence identity with the rat Na+ channel NaV1.4, and 73% with that of the snake Thamnophis sirtalis. The TMs are extremely well conserved (87%) with absolute conservation of S4 in all domains. The N-and C-termini as well as the cytoplasmic linkers between domains are more divergent. The D3-D4 linker containing the IFM motif is highly conserved except for Q1348E and K1350P. The DEKA-motif is also absolutely conserved as are the GGGS gating hinge and the QGFS motifs. BTX is thought to bind in the pore region, from the selectivity filter ring to the pore lining S6 TMs. We have identified two S to A mutations flanking the gating-hinge in domains 1 and 3 that may participate in toxin-insensitivity of the Phyllobates channel by impairing the binding of BTX. Supported by NIH GM68044(AMC) and GM30376(FB) and by COLCIENCIAS1106-12-13836(LF).