12 results on '"Rocio K. Finol-Urdaneta"'
Search Results
2. Modulation of Native and Recombinant GIRK1/2 Channels by Analgesic α-conotoxins
- Author
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Jeffrey R. McArthur, David J. Adams, Rocio K. Finol-Urdaneta, and Anuja R. Bony
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law ,Modulation ,Chemistry ,Analgesic ,Biophysics ,Recombinant DNA ,Pharmacology ,α conotoxin ,law.invention - Published
- 2020
3. Probing Voltage-Dependent Structural Changes of the VSD in Mammalian Nav with LRET
- Author
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Thomas Durek, Ana M. Correa, Tomoya Kubota, Francisco Bezanilla, Rocio K. Finol-Urdaneta, David J. Craik, and Robert J. French
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Crystallography ,biology ,Chemistry ,Sodium channel ,Energy transfer ,Voltage sensing ,Biophysics ,Xenopus ,Gating ,Conotoxin ,biology.organism_classification ,Transmembrane protein - Abstract
Voltage-gated sodium channels (Nav) play an essential role in the generation and propagation of action potentials. Mammalian Nav alpha subunits are large single polypeptide chains organized in four different domains (DI-DIV). Each domain is composed of 6 transmembrane segments (S1-S6) in which S1-S4 constitute the voltage sensing domain (VSD) and S5 and S6 form the pore. Although Nav function has been studied comprehensively, the precise structural basis for the gating mechanisms has not been fully elucidated. Recently, the crystal structures of several prokaryotic sodium channels have been solved, but they are homo-tetrameric proteins in contrast to the mammalian Navs and these studies only provide a single snapshot of the channels in one of many possible conformational states. Therefore, techniques that provide dynamic structural information are needed. In this work we have used Lanthanide-based resonance energy transfer (LRET) to obtain structural information and conformational dynamics of the VSD from each domain of the rat skeletal muscle sodium channel (Nav1.4). We prepared Nav1.4 constructs with a genetically encoded lanthanide binding tag (LBT), which binds a lanthanide (Tb3+) ion with high affinity, inserted in several strategic places at the top of the S4 segment in each domain. Two new conotoxin analogs were synthesized to function as LRET acceptors: KIIIA-Bodipy and GIIIA-Bodipy. We calculated multiple distances from each one of the domains to the pore region where the labeled toxin binds in both resting and slow inactivated states in voltage-clamped Xenopus laevis oocytes expressing our Nav1.4 constructs, which remained functionally active. The results give a geometrical map of the S4 positions of each domain in the mammalian Nav channels and provide evidence for voltage-dependent structural changes. Support: 13POST14800031 (AHA), MOP-10053 (CIHR), GM68044-07, U54GM087519 and GM030376.
- Published
- 2016
- Full Text
- View/download PDF
4. Biochemical Characterization of κM-RIIIJ, a Kv1.2 Channel Blocker
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Baldomero M. Olivera, Rocio K. Finol-Urdaneta, Andreas Dendorfer, Ping Chen, and Heinrich Terlau
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Cardioprotection ,Conus radiatus ,biology ,Chemistry ,Venom ,Cell Biology ,Pharmacology ,biology.organism_classification ,complex mixtures ,Biochemistry ,Potassium channel ,nervous system ,In vivo ,Conus ,Conus Snail ,Conotoxin ,Molecular Biology - Abstract
Conus snail (Conus) venoms are a valuable source of pharmacologically active compounds; some of the peptide toxin families from the snail venoms are known to interact with potassium channels. We report the purification, synthesis, and characterization of κM-conotoxin RIIIJ from the venom of a fish-hunting species, Conus radiatus. This conopeptide, like a previously characterized peptide in the same family, κM-RIIIK, inhibits the homotetrameric human Kv1.2 channels. When tested in Xenopus oocytes, κM-RIIIJ has an order of magnitude higher affinity (IC50 = 33 nm) to Kv1.2 than κM-RIIIK (IC50 = 352 nm). Chimeras of RIIIK and RIIIJ tested on the human Kv1.2 channels revealed that Lys-9 from κM-RIIIJ is a determinant of its higher potency against hKv1.2. However, when compared in a model of ischemia/reperfusion, κM-RIIIK (100 μg/kg of body weight), administered just before reperfusion, significantly reduces the infarct size in rat hearts in vivo without influencing hemodynamics, providing a potential compound for cardioprotective therapeutics. In contrast, κM-RIIIJ does not exert any detectable cardioprotective effect. κM-RIIIJ shows more potency for Kv1.2-Kv1.5 and Kv1.2-Kv1.6 heterodimers than κM-RIIIK, whereas the affinity of κM-RIIIK to Kv1.2-Kv1.7 heterodimeric channels is higher (IC50 = 680 nm) than that of κM-RIIIJ (IC50 = 3.15 μm). Thus, the cardioprotection seems to correlate to antagonism to heteromultimeric channels, involving the Kv1.2 α-subunit rather than antagonism to Kv1.2 homotetramers. Furthermore, κM-RIIIK and κM-RIIIJ provide a valuable set of probes for understanding the underlying mechanism of cardioprotection.
- Published
- 2010
5. Modulation of KvAP Unitary Conductance and Gating by 1-Alkanols and Other Surface Active Agents
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Jeffrey R. McArthur, Rocio K. Finol-Urdaneta, Robert J. French, Peter F. Juranka, and Catherine E. Morris
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Lipid Bilayers ,Biophysics ,Analytical chemistry ,Gating ,Decane ,Surface-Active Agents ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Pressure ,Channels and Transporters ,Lipid bilayer ,Ion channel ,Anesthetics ,030304 developmental biology ,0303 health sciences ,Ethanol ,Chemistry ,Bilayer ,Electric Conductivity ,Conductance ,Raft ,Kinetics ,Cholesterol ,Membrane ,Potassium Channels, Voltage-Gated ,Ion Channel Gating ,030217 neurology & neurosurgery - Abstract
The actions of alcohols and anesthetics on ion channels are poorly understood. Controversy continues about whether bilayer restructuring is relevant to the modulatory effects of these surface active agents (SAAs). Some voltage-gated K channels (Kv), but not KvAP, have putative low affinity alcohol-binding sites, and because KvAP structures have been determined in bilayers, KvAP could offer insights into the contribution of bilayer mechanics to SAA actions. We monitored KvAP unitary conductance and macroscopic activation and inactivation kinetics in PE:PG/decane bilayers with and without exposure to classic SAAs (short-chain 1-alkanols, cholesterol, and selected anesthetics: halothane, isoflurane, chloroform). At levels that did not measurably alter membrane specific capacitance, alkanols caused functional changes in KvAP behavior including lowered unitary conductance, modified kinetics, and shifted voltage dependence for activation. A simple explanation is that the site of SAA action on KvAP is its entire lateral interface with the PE:PG/decane bilayer, with SAA-induced changes in surface tension and bilayer packing order combining to modulate the shape and stability of various conformations. The KvAP structural adjustment to diverse bilayer pressure profiles has implications for understanding desirable and undesirable actions of SAA-like drugs and, broadly, predicts that channel gating, conductance and pharmacology may differ when membrane packing order differs, as in raft versus nonraft domains.
- Published
- 2010
6. Functional Modification of Bacterial Voltage-Gated Sodium Channels by Batrachotoxin
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Robert J. French, Jeffrey R. McArthur, Rocio K. Finol-Urdaneta, and Rachelle Gaudet
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Chemistry ,Sodium channel ,Biophysics ,Gating ,complex mixtures ,chemistry.chemical_compound ,Membrane ,Biochemistry ,Cytoplasm ,Excitatory postsynaptic potential ,Batrachotoxin ,Binding site ,Receptor - Abstract
Batrachotoxin (BTX) is an excitatory component in the skin secretions of dendrobatid frogs, which advertise their armament with the gaudy colors of their lethal skin. BTX is a hydrophobic alkaloid, which crosses cell membranes and activates voltage-gated sodium (Nav) channels of muscle, nerve and heart. BTX binding causes a shift of the activation curve toward more negative voltages, and suppresses fast and slow inactivation resulting in a massive increase in excitability of the tissue and ultimately causing paralysis. BTX is thought to act by binding in the cytoplasmic cavity of the ion-conducting pore, of pseudo-symmetric eukaryotic Nav channels. This binding site is near, or overlapping, receptor sites for amphiphylic inhibitor drugs including local anesthetics, anticonvulsants and anti-arrhythmics.The four homologous domains of eukaryotic Navs parallel the homo-tetrameric arrangement of BacNav channels, which have yielded high-resolution crystal structures. Despite the absence of a “ball-and chain” or “hinged-lid” fast inactivation mechanism, BacNavs display a “pore based” inactivation, that in some cases is quite rapid. Thus, they offer an opportunity to explore BTX actions on a molecular system of gating that is amenable to structural verification.The BacNav channels NaChBac and NavSp1 both show dramatic modification by BTX, with negative shifts of activation and inhibition of inactivation. These actions are enhanced by phenylalanine substitutions of pore domain (S6) residues, at positions which align with Phe residues that are important for BTX activation in eukaryotic Nav channels.
- Published
- 2016
7. Bases of Sodium Channel Selectivity Among Organic Cations
- Author
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Yibo Wang, Rocio K. Finol-Urdaneta, Robert J. French, and Sergei Y. Noskov
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Tetramethylammonium ,chemistry.chemical_compound ,Crystallography ,Molecular dynamics ,chemistry ,Inorganic chemistry ,Biophysics ,Moiety ,Ammonium ,Selectivity ,Alkali metal ,Ion ,Methyl group - Abstract
Hille's (1971) seminal study of the organic cation selectivity of voltage-gated sodium channels, showed a striking, sharp size cut-off for ion permeation. No ion species with an aliphatic moiety having at least the diameter of a methyl group was measurably permeant. Here, we show that, despite similar alkali cation permeability sequences for pro- and eu-karyotic channels (Finol-Urdaneta et al 2014 J Gen Physiol), relative permeabilities among organic ions (ammonium, hydrazinium, guanidinium and tetramethylammonium) are different. Using molecular dynamics simulations, we have asked to what extent 1d-pmfs based on the NavAb crystal structure might suggest a basis for NavBac selectivity among organic ions. Unlike eukaryotic channels, NavBac permeabilities do not show a size cut-off based on minimal cross-section, but are correlated with apparent binding energies in 1-dimensional potentials of mean force from simulations of single ions moving through the channel. In contrast, earlier analyses for eukaryotic Nav1s, suggest that differences among ion entry barriers are sufficient to account for relative permeabilities (Hille 1975 J Gen Physiol). We surmise that multi-ion occupancy and structural flexibility of Bac-Navs may also contribute to the observed and simulated functional differences. Meanwhile, we examined the consistency of our PMFs based on Zhu and Hummer's paper (Zhu & Hummer, 2011 J Comput Chem). The results indicate that it needs at least 10ns for particular windows to get accurate PMFs even for small molecules like ammonium.
- Published
- 2016
8. Kv1.7 - Interactions with Protons and a blocking Conotoxin
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Stefan Becker, Heinrich Terlau, Robert J. French, and Rocio K. Finol-Urdaneta
- Subjects
Stereochemistry ,Chemistry ,Toxin ,medicine ,Biophysics ,Conductance ,Protonation ,Depolarization ,Conotoxin ,Patch clamp ,Inhibitory postsynaptic potential ,medicine.disease_cause ,Potassium channel - Abstract
We have examined the interactions of protons and an inhibitory, poly-cationic conotoxin with the human voltage-gated potassium channel, hKv1.7. This channel differs from some members of the Kv1 sub family by having a titratable histidine residue near the N-terminal end of the putative pore-supporting P-helix, suggesting that intrinsic channel functions and pharmacology may depend on the pH of the external solution. Channels were expressed in HEK-293 cells and studied by whole-cell patch clamp. The voltage dependence of channel activation was evaluated using a tail-current protocol in which the voltage was stepped to -40 mV following a variable activating pre-pulse. Lowering of the pH of the external solution from 7.4 to 5.0 produced a positive shift in the half-activation of 36±3 mV (n=4). The lowering of pH also dramatically decreased the ability of the conotoxin to inhibit currents through the channels. Thus, following the largest depolarization, at pH5.0 very little inhibition was observed at a toxin concentration near IC50 for pH7.4. The tail current in the presence of the conotoxin was near the value seen in the absence of the toxin for external pH of 7.4. Our observations are consistent with a two-fold action of the conotoxin. First, they suggest that the positively charged toxin binds close enough to the S4 segment of the voltage sensor to inhibit activation following a depolarizing voltage step. Second, the observed decrease in maximal conductance at pH 7.4 following addition of the toxin is consistent with a block of current through the open channels. Toxin binding appears to be inhibited by protonation of a residue on the external surface of the channel, perhaps the histidine residue near the N-terminal end of the pore helix.
- Published
- 2009
- Full Text
- View/download PDF
9. 'Use Dependence' Without a Ball and Chain - Inhibition of Bacterial Sodium Channels by μ-Contoxins
- Author
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Robert J. French, Rocio K. Finol-Urdaneta, and Denys McMaster
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Chemistry ,Sodium channel ,Use dependence ,Biophysics ,Peak current ,Potency ,Depolarization ,Pharmacology ,Inflammatory pain ,Highly selective - Abstract
“Use-” or “state-” dependent block of voltage-gated sodium channels is characteristic of therapeutic agents used to moderate electrical activity in pathologically hyperactive tissues and combat certain forms of epilepsy, cardiac arrhythmia, myotonia, and pain. Externally acting Na channel pore blockers are not commonly used as therapeutic drugs, but intraperitoneal μ-conotoxin (μCTX) KIIIA can act as an effective analgesic against inflammatory pain in mice, at concentrations that do not cause obvious systemic side effects (Zhang et al 2007 J Biol Chem 42:30699). This action appears to result from highly specific targeting to neuronal Na channels, without essential reliance on the use-dependent potency that contributes to the efficacy of clinical local anesthetics. μCTXs, although highly selective among closely related eukaryotic Na channels, have recently been found to block bacterial Nav channels with high affinity (Chen & Chung 2012 Biophys J 102:483; Finol-Urdaneta et al 2013 Biophys J 104:136a). In addition, the block was potentiated at more depolarized holding potentials. Here, we explore the dependence of μCTX PIIIA block of NaChBac and NavSp1 on varying patterns of conditioning depolarization. Briefly, in the low picomolar range, PIIIA application was associated with faster inactivation decay during a test depolarization, and block of the peak current. The speeding of inactivation precedes the block of current following PIIIA application, and this kinetic effect was obvious at more negative holding potentials than was the decrease in peak current. In NaChBac, both activation and inactivation were shifted in the hyperpolarizing direction by ∼25-30 mV in the presence of 5pM PIIIA. The data suggest that PIIIA binding is conformation dependent, with high affinity for non-conducting, depolarization-induced states. At present, the relative importance of “pre-activated” and “in-activated” states in potentiating inhibition by PIIIA is unclear.
- Published
- 2015
10. Polymodal, High Affinity Actions of μ-Conotoxins on a Bacterial Voltage-Gated Sodium Channel
- Author
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Jeffrey R. McArthur, Rocio K. Finol-Urdaneta, Robert Glavica, and Robert J. French
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Gene isoform ,0303 health sciences ,Chemistry ,Stereochemistry ,Sodium channel ,Voltage clamp ,Sodium ,030302 biochemistry & molecular biology ,Biophysics ,Wild type ,chemistry.chemical_element ,Gating ,03 medical and health sciences ,NAV1 ,Conotoxin ,030304 developmental biology - Abstract
μ-Conotoxins (μCTXs) are potent blockers of eukaryotic sodium channels, and individual members of the μCTX family are highly specific for particular Nav1 isoforms. We have begun to explore μCTX interactions with NaChBac, a prokaryotic Nav channel closely related to NavAb, whose crystal structure was recently determined (Payandeh et al, 2011, Nature 475:353). Our data reveal actions on both ion conduction and gating, consistent with polymodal actions on the pore domain.Under voltage clamp, whole-cell currents from NaChBac expressed in tsA-201 cells were reduced, up to nearly complete block, by concentrations in the pM to μM range, for wildtype toxins and derivatives of μCTX PIIIA and KIIIA. For wildtype PIIIA, dose-response data (0.001-10,000nM) yielded the following: IC50=0.005nM; maximal fraction of current blocked = 0.95; Hill coefficient = 0.7. Chen & Chung (2012, Biophys. J. 102:483) predicted an IC50 of 0.1nM for PIIIA when the NavAb channel is occupied by 2 sodium ions.Even at very low [PIIIA] (1pM), the unblocked currents showed increasing rates of inactivation as the peptides were washed in, suggesting a gating modulation, that was not tightly associated with pore block. For μCTX PIIIA (0.1nM), or KIIIA (30nM), inactivation accelerated by ∼10-fold. Substitution of key basic residues (PIIIA-R14A and KIIIA-K7A) reduced blocking potency and decreased the speeding of inactivation to less than 2-fold.Given the remarkably high selectivity and affinity that μCTXs show for certain eukaryotic Nav channels with highly asymmetric pores, it may seem surprising that they bind to symmetric bacterial Nav channels with such high affinity. However, in both cases, there is the potential for multiple electrostatic interactions to sum and yield potent binding. Our data suggest that pore-block, and acceleration of inactivation, result from distinct molecular actions of the toxin.
- Published
- 2013
11. Monovalent Ion Selectivity of the Homotetrameric Bacterial Na Channel, NaChBac
- Author
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Sergei Y. Noskov, Rocio K. Finol-Urdaneta, Robert J. French, and Ahmed Al-Sabi
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chemistry.chemical_compound ,Crystallography ,Monomer ,chemistry ,Stereochemistry ,Biophysics ,Ammonium ,Context (language use) ,Crystal structure ,Selectivity ,Reversal potential ,Alkali metal ,Ion - Abstract
NaChBac, from Bacillus halodurans, is a homotetrameric Nav channel, with each monomer having 6 putative transmembrane helices. Its selectivity filter appears to be lined by a ring of 4 glutamate residues (E191 from each monomer). We expressed NaChBac channels in HEK293 cells and studied their selectivity to monovalent organic and alkali cations. Reversal potential shifts were determined from whole-cell currents, following substitution of a test cation for extracellular Na+, and relative permeabilities (PX/PNa) were calculated using the Goldman-Hodgkin-Katz equation. Reversal potentials (Erev) were routinely measured from plots of peak INa vs V, but for ions showing small inward currents, values were checked against tail currents, measured immediately after a maximally activating prepulse. Among the alkali cations, only Na+, Li+ and K+ were measurably permeant. Of 14 organic cations, at pH 7.4, only hydrazinium (HZ+) was measurably permeant (PX/PNa, ≈ 0.8, 0.3, 0.7 respectively, for X= Li, K, HZ
- Published
- 2012
12. Tracking Voltage-Dependent Conformational Changes of the VSD in Nav with LRET
- Author
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Stephen B. H. Kent, Tomoya Kubota, Pedro Brugarolas, Francisco Bezanilla, Ana M. Correa, Robert J. French, Rocio K. Finol-Urdaneta, Bobo Dang, and Ludivine Frezza
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Tityus serrulatus ,Förster resonance energy transfer ,Scorpion toxin ,biology ,Chemistry ,Stereochemistry ,Sodium channel ,Biophysics ,Xenopus ,Gating ,biology.organism_classification ,Small molecule ,Transmembrane protein - Abstract
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.
- Published
- 2014
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