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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, 2010
    Co-Authors: Ludivine Frezza, Santiago Castaño, Leonardo Fierro, Francisco Bezanilla, Helberg Asencio, Walter Sandtner, Ana M. Correa
    Abstract:

    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, 2009
    Co-Authors: Santiago Castaño, Ludivine Frezza, Alain J. Labro, Leonardo Fierro, Francisco Bezanilla, Ana M. Correa
    Abstract:

    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, 2009
    Co-Authors: Santiago Castaño, Ludivine Frezza, Alain J. Labro, Leonardo Fierro, Francisco Bezanilla, Ana M. Correa
    Abstract:

    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, 2010
    Co-Authors: Ludivine Frezza, Santiago Castaño, Leonardo Fierro, Francisco Bezanilla, Helberg Asencio, Walter Sandtner, Ana M. Correa
    Abstract:

    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, 2009
    Co-Authors: Santiago Castaño, Ludivine Frezza, Alain J. Labro, Leonardo Fierro, Francisco Bezanilla, Ana M. Correa
    Abstract:

    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, 2009
    Co-Authors: Santiago Castaño, Ludivine Frezza, Alain J. Labro, Leonardo Fierro, Francisco Bezanilla, Ana M. Correa
    Abstract:

    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).

Ian J. Wang - One of the best experts on this subject based on the ideXlab platform.

  • Inversely related aposematic traits: reduced conspicuousness evolves with increased toxicity in a polymorphic Poison-Dart Frog.
    Evolution; international journal of organic evolution, 2011
    Co-Authors: Ian J. Wang
    Abstract:

    Prevailing theory contends that aposematic coloration evolves in tandem with toxicity so that the evolution of increased toxicity will accompany the evolution of greater conspicuousness. Although variation in aposematic coloration within single species should be selectively constrained, because individuals varying from a predator-recognized warning signal will incur greater risk of predation, several species of Poison-Dart Frogs display remarkable phenotypic variation. This variation may have evolved to match different levels of toxicity, and these species provide excellent opportunities to examine the evolution of aposematic coloration. Here, I test whether increased conspicuousness in the granular Poison-Dart Frog evolved in tandem with increased toxicity. Contrary to classical predictions, toxicity assays, spectral reflectance measurements, and phylogenetic reconstruction reveal that the less conspicuous color morphs are actually significantly more toxic than the brightest, most conspicuous phenotypes and that the more toxic, less-conspicuous form evolved from a less toxic, more conspicuous ancestor. Through gas chromatography--mass spectrometry analysis of toxin profiles, I traced the increase in toxicity in the less-conspicuous populations to an acquisition of specific alkaloids, some of which are proven convulsants. These results challenge the tenet that increased conspicuousness always evolves with increased toxicity and support the idea that once aposematism has been established in a species, phenotypic variation may evolve from brightness and toxicity becoming decoupled.

  • genetic structure is correlated with phenotypic divergence rather than geographic isolation in the highly polymorphic strawberry Poison Dart Frog
    Molecular Ecology, 2010
    Co-Authors: Ian J. Wang, Kyle Summers
    Abstract:

    : Phenotypic and genetic divergence can be influenced by a variety of factors, including sexual and natural selection, genetic drift and geographic isolation. Investigating the roles of these factors in natural systems can provide insight into the relative influences of allopatric and ecological modes of biological diversification in nature. The strawberry Poison Frog, Dendrobates pumilio, presents an excellent opportunity for this kind of research, displaying a diverse array of colour morphs and inhabiting a heterogeneous landscape that includes oceanic islands, fragmented rainforest patches and wide expanses of suitable habitat. In this study, we use 15 highly polymorphic microsatellite loci to estimate population structure and gene flow among populations from across the range of D. pumilio and a causal modelling framework to statistically test 12 hypotheses regarding the geographic and phenotypic variables that explain genetic differentiation within this system. Our results demonstrate that the genetic distance between populations is most strongly associated with differences in dorsal coloration. Previous experimental studies have shown that phenotypic differences can result in sexual and natural selection against non-native phenotypes, and our results now show that these forces lead to genetic isolation between different colour morphs in the wild, presenting a potential case of incipient speciation through selection.

  • Genetic structure is correlated with phenotypic divergence rather than geographic isolation in the highly polymorphic strawberry Poison‐Dart Frog
    Molecular ecology, 2009
    Co-Authors: Ian J. Wang, Kyle Summers
    Abstract:

    Phenotypic and genetic divergence can be influenced by a variety of factors, including sexual and natural selection, genetic drift and geographic isolation. Investigating the roles of these factors in natural systems can provide insight into the relative influences of allopatric and ecological modes of biological diversification in nature. The strawberry Poison Frog, Dendrobates pumilio, presents an excellent opportunity for this kind of research, displaying a diverse array of colour morphs and inhabiting a heterogeneous landscape that includes oceanic islands, fragmented rainforest patches and wide expanses of suitable habitat. In this study, we use 15 highly polymorphic microsatellite loci to estimate population structure and gene flow among populations from across the range of D. pumilio and a causal modelling framework to statistically test 12 hypotheses regarding the geographic and phenotypic variables that explain genetic differentiation within this system. Our results demonstrate that the genetic distance between populations is most strongly associated with differences in dorsal coloration. Previous experimental studies have shown that phenotypic differences can result in sexual and natural selection against non-native phenotypes, and our results now show that these forces lead to genetic isolation between different colour morphs in the wild, presenting a potential case of incipient speciation through selection.

  • Highly polymorphic microsatellite markers for the highly polymorphic strawberry Poison-Dart Frog and some of its congeners
    Conservation Genetics, 2009
    Co-Authors: Ian J. Wang, Kyle Summers
    Abstract:

    Members of the Poison-Dart Frog genus Oophaga, including the strawberry Poison-Dart Frog (O. pumilio) display among the most striking examples of color polymorphism of any amphibians. We developed twelve novel microsatellite markers with di-, tri-, and tetra-nucleotide repeats for this genus. These loci are highly polymorphic with between 2 and 29 alleles (average = 14.7) and high heterozygosity (H O = 0.704). These highly polymorphic markers should be useful for resolving fine-scale genetic differences between the different color morphs of these highly variable species, for investigations into the ecological importance of this variation, and for determining the effects of habitat loss and fragmentation on population persistence in these species.

  • RAPID COLOR EVOLUTION IN AN APOSEMATIC SPECIES: A PHYLOGENETIC ANALYSIS OF COLOR VARIATION IN THE STRIKINGLY POLYMORPHIC STRAWBERRY Poison-Dart Frog
    Evolution; international journal of organic evolution, 2008
    Co-Authors: Ian J. Wang, H. Bradley Shaffer
    Abstract:

    Aposematism is one of the great mysteries of evolutionary biology. The evolution of aposematic coloration is poorly understood, but even less understood is the evolution of polymorphism in aposematic signals. Here, we use a phylogeographic approach to investigate the evolution of color polymorphism in Dendrobates pumilio, a well-known Poison-Dart Frog (family Dendrobatidae), which displays perhaps the most striking color variation of any aposematic species. With over a dozen color morphs, ranging from bright red to dull green, D. pumilio provides an ideal opportunity to examine the evolution of color polymorphism and evolutionary shifts to cryptic coloration in an otherwise aposematic species. We constructed a phylogenetic tree for all D. pumilio color morphs from 3051bp of mtDNA sequence data, reconstructed ancestral states using parsimony and Bayesian methods, and tested the recovered tree against constraint trees using parametric bootstrapping to determine the number of changes to each color type. We find strong evidence for nearly maximal numbers of changes in all color traits, including five independent shifts to dull dorsal coloration. Our results indicate that shifts in coloration in aposematic species may occur more regularly than predicted and that convergence in coloration may indicate that similar forces are repeatedly driving these shifts.

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, 2017
    Co-Authors: Sho-ya Wang, Ging Kuo Wang
    Abstract:

    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, 2017
    Co-Authors: Sho-ya Wang, Ging Kuo Wang
    Abstract:

    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, 2010
    Co-Authors: Ludivine Frezza, Santiago Castaño, Leonardo Fierro, Francisco Bezanilla, Helberg Asencio, Walter Sandtner, Ana M. Correa
    Abstract:

    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, 2009
    Co-Authors: Santiago Castaño, Ludivine Frezza, Alain J. Labro, Leonardo Fierro, Francisco Bezanilla, Ana M. Correa
    Abstract:

    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, 2009
    Co-Authors: Santiago Castaño, Ludivine Frezza, Alain J. Labro, Leonardo Fierro, Francisco Bezanilla, Ana M. Correa
    Abstract:

    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).