Voltage-gated sodium channels (Nav) are fundamental for the generation and the propagation of action potentials. Mammalian Nav alpha subunits are single macromolecules organized in four different domains (DI-DIV). Each is composed of 6 transmembrane segments (S1-S6) from which S1-S4 constitute the voltage sensing domain (VSD) and with S5 and S6 constituting the pore. While Nav function has been studied extensively, the exact structural mechanisms of gating are not fully understood. Recently, the crystal structure of the prokaryotic sodium channel, NavAb, has been solved, but NavAb is a homotetrameric protein in contract to the mammalian Navs. Thus many questions were not answered by the prokaryotic channel structures. To resolve the voltage dependent conformational changes of Nav, we tracked conformational changes of the VSD from each domain of the rat skeletal muscle sodium channel (Nav1.4) using Lanthanide-based Resonance Energy Transfer (LRET), a FRET technique that allows for precise measurement of intermolecular distances by taking advantage of the special properties of lanthanide as an energy donor. We prepared Nav1.4 constructs with a genetically encoded lanthanide binding tag (LBT), which holds a lanthanide (Tb3+) ion with high affinity, inserted at the top of the S4 segment in each domain. Also, we synthesized two toxins conjugated to dyes to function as acceptors: the pore-blocking small molecule tetrodotoxin conjugated with a HiLyte fluor488 (TTX-F), and the peptide β scorpion toxin Ts1, from the Brazilian scorpion Tityus serrulatus, conjugated with Alexa488 (Ts1-Alexa488). Having several donor positions (Tb3+ ions in LBT's) and two different acceptor positions (TTX-F and Ts1-Alexa488), we calculated multiple distances in voltage-clamped Xenopus laevis oocytes expressing our Nav1.4 constructs that remained functionally active. The results provide new insight to structure-function information in mammalian Nav channels. Support: 13POST14800031 (AHA), MOP-10053 (CIHR), GM68044-07, U54GM087519 and GM030376.

Magnesium (Mg2+) plays a central role in biology, regulating the activity of many enzymes and stabilizing the structure of key macromolecules. In bacteria, CorA is the primary source of Mg2+ uptake and is self-regulated by intracellular Mg2+. Using a gating mutant at the divalent ion binding site, we were able to characterize CorA selectivity and permeation properties to both monovalent and divalent cations under perfused two-electrode voltage clamp. The present data demonstrate that under physiological conditions, CorA is a multioccupancy Mg2+-selective channel, fully excluding monovalent cations, and Ca2+, whereas in absence of Mg2+, CorA is essentially nonselective, displaying only mild preference against other divalents (Ca2+ > Mn2+ > Co2+ > Mg2+ > Ni2+). Selectivity against monovalent cations takes place via Mg2+ binding at a high-affinity site, formed by the Gly-Met-Asn signature sequence (Gly312 and Asn314) at the extracellular side of the pore. This mechanism is reminiscent of repulsion models proposed for Ca2+ channel selectivity despite differences in sequence and overall structure.

Single-channel recordings revolutionized our understanding of voltage-gated ion channels by allowing observation of behaviors that are obscured by large ensemble averages. Yet despite its power, single-channel recordings only allow indirect inference about the motions of the voltage sensor since only transitions between "open" and "closed" states of the channel can be seen; most transitions between states remain hidden. For this reason, direct measurement of the motion of a single voltage sensor has long been a goal to understand the details of voltage sensing; unfortunately, at present the elementary charge transition is below experimental resolution. We report here observation of fluorescence from single voltage sensors conjugated to fluorescent proteins. These recordings respond to voltage and are able to recapitulate macroscopic recordings when averaged together. The protein we used is the "ArcLight" voltage sensor (Jin, L. et al. Neuron, 2012.), along with derivatives thereof. This sensor consists of the voltage sensing domain from Ci-VSP coupled to a GFP derivative, and it shows robust changes in fluorescence in response to voltage. Our recordings are taken from oocyte membranes using total internal reflection microscopy at a frame rate of 500 hertz and at a temperature of approximately 13 degrees Celsius. This combination of low temperature and fast acquisition allows detection of residencies of the protein at distinct fluorescence levels with stochastic movement between these levels being biased by voltage. Presumably these distinct fluorescence levels correspond to distinct states of the voltage sensor. The transitions between these states can be analyzed and modeled, producing a novel picture of how the voltage sensor moves and how these movements are influenced by membrane potential. Support: NIH GM030376.

Mammalian voltage-gated sodium channels (Nav) are composed of two subunits: a monomeric pore-forming subunit (α-subunit), that contains four domains (DI-DIV), each composed of a voltage-sensing domain (VSD, S1-S4) and a pore domain (S5-S6) and auxiliary subunits (β-subunits) that are transmembrane proteins with type I topology: containing an extracellular amino-terminus, a single transmembrane segment and an intracellular carboxyl terminus. A large body of literature has shown that interaction between α- and β-subunits results in Nav with altered gating kinetics suggesting that β-subunits may directly interact with VSD. However, aspects of the stoichiometry, arrangement and molecular interaction between α and β subunits remain unclear. In this study, we explored the location of β1 in relation to the rat skeletal muscle sodium channel α-subunit (Nav1.4) using lanthanide-based resonance energy transfer (LRET) via two strategies. 1) Four Nav1.4 constructs were designed to encode a Tb3+ binding-tag (Nav1.4-LBT) on top of the S4 of each domain (DI-LBT, DII-LBT, DIII-LBT and DIV-LBT) as energy donor. A hexa-histidine-tag was inserted in β1 (6His-β1), which binds a Cu2+ ion to act as acceptor. 2) β1 constructs were designed to encode at an extracellular site an LBT that binds Tb3+ as energy donor while the acceptor was Alexa488 conjugated to Ts1, a β scorpion toxin which binds to DII-VSD in Nav1.4. For both experiments, we used Xenopus laevis oocytes co-expressing Nav1.4 and β1 constructs injected with the cRNA at 1:1 molar ratio. In preliminary results, DIV-LBT + 6His-β1 oocytes displayed robust energy transfer between Tb3+ and Cu2+, indicating that at least one β1-subunit is located at ∼28 Å of S4-DIV. Support: 13POST14800031 (AHA), MOP-10053 (CIHR), GM68044-07, U54GM087519 and GM030376.

In nerves and muscles, action potentials are initiated by the rapid activation of voltage-gated sodium (Nav) channels and terminated by the delayed activation of voltage-gated potassium (Kv) channels. This sequential activation, which is the prerequisite for the genesis of the action potential, requires faster activation kinetics of the voltage-sensor domains (VSD) in Nav as compared to Kv channels. Despite on decades of investigations, the molecular determinants and mechanisms underlying this phenomenon remain elusive. Here, we show that this differential gating is mostly imparted by six conserved hydrophilic residues Thr or Ser located in the S2 and S4 segments of the VSD in domains I-III of Nav channels while these positions are commonly occupied by hydrophobic residues in the VSD of Kv channels. Hydrophilic substitutions at the S2 position in the Shaker Kv channel accelerate the gating charge transfer by decreasing its energy barrier while hydrophilic substitutions at the S4 position speed up VSD activation by destabilizing its resting conformation. Interestingly, these hydrophilic residues are present in a Nav-related gene expressed in an evolutionary-distant unicellular choanoflagellate, suggesting that rapidly-gated Nav channels evolved before the emergence of metazoans and their nervous systems. We also show that the physiological co-expression of the ubiquitous regulatory β1 subunit further accelerates VSD movement in both a neuronal and a muscular Nav, providing a molecular basis for the β1-dependent fast gating mode previously detected from the ionic conductance. Our study uncovers the fundamental molecular determinants and possible mechanisms that enabled the differential gating in sodium and potassium channels and the emergence of the action potential. This work was supported by NIH grant GM030376.

Most action potentials are produced by the sequential activation of voltage-gated sodium (Nav) and potassium (Kv) channels. This is mainly achieved by the rapid conformational rearrangement of voltage-sensor (VS) modules in Nav channels, with activation kinetics up to 6-fold faster than Shaker-type Kv channels. Here, using mutagenesis and gating current measurements, we show that a 3-fold acceleration of the VS kinetics in Nav versus Shaker Kv channels is produced by the hydrophilicity of two “speed-control” residues located in the S2 and S4 segments in Nav domains I–III. An additional 2-fold acceleration of the Nav VS kinetics is provided by the coexpression of the β1 subunit, ubiquitously found in mammal tissues. This study uncovers the molecular bases responsible for the differential activation of Nav versus Kv channels, a fundamental prerequisite for the genesis of action potentials.

Voltage control over enzymatic activity in voltage-sensitive phosphatases (VSPs) is conferred by a voltage-sensing domain (VSD) located in the N terminus. These VSDs are constituted by four putative transmembrane segments (S1 to S4) resembling those found in voltage-gated ion channels. The putative fourth segment (S4) of the VSD contains positive residues that likely function as voltage-sensing elements. To study in detail how these residues sense the plasma membrane potential, we have focused on five arginines in the S4 segment of the Ciona intestinalis VSP (Ci-VSP). After implementing a histidine scan, here we show that four arginine-to-histidine mutants, namely R223H to R232H, mediate voltage-dependent proton translocation across the membrane, indicating that these residues transit through the hydrophobic core of Ci-VSP as a function of the membrane potential. These observations indicate that the charges carried by these residues are sensing charges. Furthermore, our results also show that the electrical field in VSPs is focused in a narrow hydrophobic region that separates the extracellular and intracellular space and constitutes the energy barrier for charge crossing.

In voltage-sensitive phosphatases (VSPs), a transmembrane voltage sensor domain (VSD) controls an intracellular phosphoinositide phosphatase domain, thereby enabling immediate initiation of intracellular signals by membrane depolarization. The existence of such a mechanism in mammals has remained elusive, despite the presence of VSP-homologous proteins in mammalian cells, in particular in sperm precursor cells. Here we demonstrate activation of a human VSP (hVSP1/TPIP) by an intramolecular switch. By engineering a chimeric hVSP1 with enhanced plasma membrane targeting containing the VSD of a prototypic invertebrate VSP, we show that hVSP1 is a phosphoinositide-5-phosphatase whose predominant substrate is PI(4,5)P2. In the chimera, enzymatic activity is controlled by membrane potential via hVSP1’s endogenous phosphoinositide binding motif. These findings suggest that the endogenous VSD of hVSP1 is a control module that initiates signaling through the phosphatase domain and indicate a role for VSP-mediated phosphoinositide signaling in mammals.

Phosphoinositide (PI) concentrations in cell membranes play an important role in many cellular processes. PI phosphatases are crucial for the regulation of these concentrations. Among these phosphatases are the voltage-sensitive phosphatases (VSPs) like the prototypical Ci-VSP, which provide a new paradigm for the control of enzymatic activity. Recently, we showed that voltage sensitivity can be conferred to the cytoplasmic phosphatase PTEN: by fusing the voltage sensor domain (VSD) of Ci-VSP to the catalytic domain (CD) of PTEN, we generated an engineered chimeric VSP, Ci-VSPTEN. Here we extend this work and report on a chimera created by fusing Ci-VSP's VSD to the CD of the putative PI-phosphatase TPIPα. We demonstrate that this Ci-VSP/TPIP chimera exhibits voltage dependent enzymatic activity. The substrate and position specificity of Ci-VSP/TPIP is analyzed using genetically encoded PI-specific fluorescence labeled probes, the membrane binding of which is assessed using total internal reflection fluorescence (TIRF) microscopy. Control over the membrane voltage is achieved by whole-cell patch clamping. We find that upon depolarization, the membrane binding of the PIP3 specific probe, Btk-PH, and the PI(4,5)P2 specific probe, PLCδ1-PH, decreases, when co-expressed with Ci-VSP/TPIP. In contrast, membrane binding of the PI(3,4)P2 specific TAPP1-PH and the PI(4)P specific OSBP-PH probes increases upon depolarization. These findings identify the Ci-VSP/TPIP chimera as a voltage-sensitive PI(4,5)P2/PIP3-5’-phosphatase. We conclude that TPIPα is a 5’-phosphatase in-vivo, in contrast to PIP3-3’-phosphatase activity in-vitro reported previously (Walker et al., Biochem. J. (2001) 360, 277 −283). These data will help to further the understanding of the mechanism by which voltage control is exerted in VSPs. Additionally, our experiments demonstrate the usefulness of engineered VSPs as a novel tool for the analysis of PI-phosphatases in-vivo, an obligatory complement to in-vitro characterization. Supported by Deutsche Forschungsgemeinschaft (SFB593 TPA12 to D.O.)

Segments S4 and S5 in Voltage Gated Channels potassium channels are contiguous and specific residues of these segments get in atomic proximity in a state-dependent way (Lainéet al., 2003; Lewis et al., 2008). In Shaker, the double mutation R362H+A419H stabilizes the conducting state of the channels when a metal bridge is formed in the presence of Zn2+ (Lainéet al., 2003). These results were obtained from ionic conduction experiments but gave no direct information on the dynamics of the Voltage Sensing Domain (VSD) of Shaker. As a proxy for the movement of the VSD, we studied the proton currents through the VSD that results by the double mutation R362H+A419H, on the ultra-fast-inactivating Shaker W434F. When the holding potential (HP) was 0 mV, the current-voltage relation of the proton current (Ip-V) was shifted towards negative potentials as compared to the Ip-V when HP was −90 mV, as expected from the relaxation that the VSD undergoes at maintained depolarization. When HP was 0 mV, the proton current was decreased and the Ip-V was further shifted by increasing the concentration of Ni2+ or Zn2+ (10 μM-100 μM). In contrast, no changes were observed in the Ip-V voltage dependence with Ni2+ or Zn2+ when holding at −90 mV. In the presence of Ni2+ or Zn2+ the proton current showed a second slower kinetic component, whose relative amplitude was increased with an increase in Ni2+or Zn2. Fluorescence recordings with a probe in M356C showed that Zn2+ decreased the rate of TMRM dequenching when pulsing to negative potentials from an HP of 0 mV, consistent with the proton current results. These observations indicate that the metal bridge between R362H and A419H stabilizes the relaxed state of the VSD (Support NIHGM030376).

In prokaryotes, Magnesium homeostasis is tightly controlled through the workings of three distinct transport systems, with CorA being the primary source of Mg2+ uptake. The recent structures of CorA from the thermophilic eubacteria Thermotoga maritima (TmCorA) have provided an excellent model for a molecular understanding of Mg2+ transport. Yet, a detailed characterization of CorA function has been conspicuously missing so far, as only in-vivo bulk assays have been available to evaluate Mg2+ translocation leaving unanswered fundamental questions related to CorA ion selectivity and specificity, gating mechanism and regulation. Here we have developed a system for high-level expression of TmCorA in Xenopus oocytes that allows for an accurate functional readout of Mg2+ transport through several electrophysiological techniques. Mg2+ currents from TmCorA in the order of 1 to 10 μA were routinely obtained with the cut-open voltage clamp technique and two electrodes Voltage clamp. We demonstrate that CorA act as a strong inward rectifier, an observation in agreement with its physiological role as the primary source of Mg2+ uptake. As expected, Mg2+ currents are blocked by Co(III) hexamine, a structural analog of a hydrated Mg2+. CorA selectivity towards Magnesium vs. other divalent and monovalents was characterized using a perfusion TEV setup. The divalent selectivity series for CorA was determined as Co2+>=Mg2+>Ni2+>Zn2+>>>Ca2+ = Ba2+. Macroscopic CorA currents record from oocytes macropatches show that Mg2+-binding to the cytoplasm domain act as a gating factor. Single channel transitions are still below our detection capabilities and efforts are ongoing to characterize the conductance of TmCorA through noise analysis. The ability to evaluate CorA function by means of electrophysiological methods will allow for a detailed analysis of structure/function relationships in CorA.

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).

In voltage-dependent ion channels, the movement of the voltage-sensing S4 helices produces gating currents. The charge displaced as a function of the membrane potential (Q-V) is well described by a sequential two-state Boltzmann relation, indicating that there are at least two steps of gating charge movement from their Resting state to the Active state. In addition, it has been shown that at a maintained positive potential, the S4 helices of voltage-gated Na, Ca and K channels and the voltage sensitive phosphatase Ci-VSP, undergo a slower secondary conformational transition stabilizing the sensor in a Relaxed (inactivated) state. From the Relaxed state, the Q-V relation exhibits a strong shift towards negative potentials when compared to the Q-V relation measured from the resting state. We engineered gating perturbations in the Shaker potassium channel, by substituting specific aromatic residues in positions spatially close to the S4. One of these mutants, in position I241 of S1, part of the hydrophobic plug of the voltage sensor, when mutated to tryptophan (I241W), produces a strong split in the Q-V when measured from the resting state. By labeling M356C with TMRM we also find the same split in the fluorescence-voltage curve. We propose that the presence of the tryptophan in the 241 position favors an interaction with one of the positively-charged arginines along the S4, thus stabilizing a fleeting intermediate state in the gating pathway. However, in the I241W mutant, the split in the Q-V almost disappears when the gating currents are measured from the relaxed state and the same result is seen with the fluorescence-voltage curve. This result and the effect of other tryptophan perturbations near the S4 segment strongly support the existence of two structurally distinct gating pathways for the movement of the S4 helices. Supported: NIHGM030376.

The Ciona intestinalis Voltage Sensor-containing Phosphatase (Ci-VSP) is a member of the Voltage Sensitive Phosphatase family that exhibits membrane potential-controlled enzymatic activity. Alignments of the amino acid sequence of Ci-VSP against Voltage Gated Channels (VGC) indicate that its Voltage Sensing Domain (VSD) is formed by four transmembrane segments. The putative fourth segment (S4) of Ci-VSP extends between the arginine (R) in position 217 and the glutamine (Q) in position 239, containing five arginines, which might be the voltage sensing charges. Although it has been shown that R229 and R232 are critical for voltage sensing in Ci-VSP (Murata et al., 2005), the role of the remaining charges is still unclear. To address this issue, we have performed a partial Histidine Scanning of the S4 of Ci-VSP, following the paradigm established for the VGC Shaker (Starace and Bezanilla, 2004). The voltage dependence of the sensing current of the R217H mutant was modulated by pH. Decreasing the external pH shifted the Q-V curve towards positive potentials, while a pH increase had the converse effect, consistent with the finding that neutralizing R217 (R217Q) produces a negative shift of the voltage dependence of Ci-VSP (Kohout et al., 2008). However, the total net charge of R217H did not change with pH, indicating that R217 does not participate in sensing the membrane potential. When the second arginine is replaced by histidine, the resultant mutant (R223H) exhibits a voltage dependent proton current which closes at positive potentials, resembling the current recorded from Shaker-W434F with its first gating charge replaced by histidine (R362H). This result strongly suggests that R223 has access to both the intra- and the extracellular media depending on voltage. Taken together, our results indicate that R223 is the most extracellularly located sensing charge of the Ci-VSP S4 segment. (Support: NIHGM030376)

1263-Pos Board B107 Cloning and Sequence Analysis of the Voltage-Gated Muscle Naþ Channel from the Poison Dart Frog Phyllobates aurotaenia Santiago Castano1,2, Ludivine Frezza1, Alain Labro3, Leonardo Fierro2, Francisco Bezanilla1, Ana M. Correa1. The University of Chicago, Chicago, IL, USA, Grupo de Biologia Integrativa, Universidad del Valle, Cali, Colombia, University of Antwerp, Wilrijk, Belgium. 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 Naþ 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 Naþ 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 Naþ 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 Naþ 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).

In Shaker K+ channels, lanthanide binding tags (LBT) were encoded in 4 consecutive positions on the top of the S4 segment and in 4 consecutive positions on the top of the S3 segment. To constrain the LBT position a truncated S3-S4 linker Shaker construct was used. Tagged channels were expressed in Xenopus laevis oocytes and LRET-based distance measurements were conducted between Tb3+ ions bound to the LBT and Bodipy-Fl attached to the pore-blocker Agitoxin-2. Distance measurements for each of the tagged Shaker constructs were repeated with 3 toxins labeled at positions D20C, Q13C and N5C, respectively. Distances were determined in the three main conformational states of the channel: closed, open and open-inactivated. Voltage-dependent K+ channels are comprised of 4 subunits, symmetrically arranged around a central pore. In our measurements each of the subunits carried a LBT. With the toxin bound to the channel pore, energy is transferred from the 4 donors on the channel to the 1 acceptor element on the toxin. Due to this geometry the lifetime of the sensitized emission decay is composed of 4 exponential components corresponding to 4 donor-acceptor distances (Posson, Selvin 2008). We could determine all 4 distances by fitting a geometrical model to the decay and also determine the positions of the bound Tb3+ ion in the LBT in x, y and z. The resultant coordinates are used to refine the models based on the crystal structure of KV1.2 for the closed, open and open-inactivated states. The most important finding of this study is that the position of the voltage sensor changes, not only when going from the closed to the open state, but also when going from the open to the open-inactivated state. Support: AHA07257632(WS), NIHGM30376(FB), NIHGM68044(AMC).

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).

study we used a selection of peptide neurotoxins against the cardiac NaV1.5 channel to demonstrate the feasibility of screening slow-binding molecules by applying sophisticated voltage protocols, and acquiring stable, high quality recording for more than 30mins. The toxins used were: 1) JingZhaotoxin-II which is a potentiator or positive modulator of NaV1.5 channels, 2) ProTx-II, which is an inhibitor of NaV1.5 channels, and 3) u-Conotoxin PIIIA a NaV1.4 inhibitor was used as a negative control. The IonWorks Barracuda Plus platform was used for these studies. Results are presented which are in good agreement with peer-reviewed publications. Taken together, these results demonstrate the high-throughput capabilities of this platform for measuring peptide toxins targeted against ion channels.