31 results on '"Batrachotoxin"'
Search Results
2. Trans-Channel Interactions in Batrachotoxin-Modified Skeletal Muscle Sodium Channels: Voltage-Dependent Block by Cytoplasmic Amines, and the Influence of μ-Conotoxin GIIIA Derivatives and Permeant Ions
- Author
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Jeffrey R. McArthur, Evgeny Pavlov, Quanli Ma, Gerald W. Zamponi, Tatiana Britvina, Iván E. Sierralta, and Robert J. French
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Cytoplasm ,Sodium ,Inorganic chemistry ,Static Electricity ,Biophysics ,chemistry.chemical_element ,Ligands ,01 natural sciences ,Sodium Channels ,Ion ,03 medical and health sciences ,chemistry.chemical_compound ,Sodium channel blocker ,0103 physical sciences ,Static electricity ,Conotoxin ,Channels, Receptors, and Electrical Signaling ,Amines ,Batrachotoxins ,Muscle, Skeletal ,030304 developmental biology ,0303 health sciences ,010304 chemical physics ,Chemistry ,Ligand ,Sodium channel ,Quaternary Ammonium Compounds ,Kinetics ,Batrachotoxin ,Conotoxins ,Protein Binding ,Sodium Channel Blockers - Abstract
External mu-conotoxins and internal amine blockers inhibit each other's block of voltage-gated sodium channels. We explore the basis of this interaction by measuring the shifts in voltage-dependence of channel inhibition by internal amines induced by two mu-conotoxin derivatives with different charge distributions and net charges. Charge changes on the toxin were made at residue 13, which is thought to penetrate most deeply into the channel, making it likely to have the strongest individual interaction with an internal charged ligand. When an R13Q or R13E molecule was bound to the channel, the voltage dependence of diethylammonium (DEA)-block shifted toward more depolarized potentials (23 mV for R13Q, and 16 mV for R13E). An electrostatic model of the repulsion between DEA and the toxin simulated these data, with a distance between residue 13 of the mu-conotoxin and the DEA-binding site of approximately 15 A. Surprisingly, for tetrapropylammonium, the shifts were only 9 mV for R13Q, and 7 mV for R13E. The smaller shifts associated with R13E, the toxin with a smaller net charge, are generally consistent with an electrostatic interaction. However, the smaller shifts observed for tetrapropylammonium than for DEA suggest that other factors must be involved. Two observations indicate that the coupling of permeant ion occupancy of the channel to blocker binding may contribute to the overall amine-toxin interaction: 1), R13Q binding decreases the apparent affinity of sodium for the conducting pore by approximately 4-fold; and 2), increasing external [Na(+)] decreases block by DEA at constant voltage. Thus, even though a number of studies suggest that sodium channels are occupied by no more than one ion most of the time, measurable coupling occurs between permeant ions and toxin or amine blockers. Such interactions likely determine, in part, the strength of trans-channel, amine-conotoxin interactions.
- Published
- 2008
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3. Trans-Channel Interactions in Batrachotoxin-Modified Rat Skeletal Muscle Sodium Channels: Kinetic Analysis of Mutual Inhibition between μ-Conotoxin GIIIA Derivatives and Amine Blockers
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Evgeny Pavlov, Gerald W. Zamponi, Tatiana Britvina, Quanli Ma, and Robert J. French
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Stereochemistry ,Kinetics ,Biophysics ,Ligands ,Binding, Competitive ,Sodium Channels ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Animals ,Channels, Receptors, and Electrical Signaling ,Conotoxin ,Amines ,Batrachotoxins ,Muscle, Skeletal ,030304 developmental biology ,0303 health sciences ,Sodium channel ,Rats ,Quaternary Ammonium Compounds ,Dissociation constant ,Membrane ,chemistry ,Batrachotoxin ,Amine gas treating ,Steady state (chemistry) ,Conotoxins ,030217 neurology & neurosurgery ,Protein Binding ,Sodium Channel Blockers - Abstract
R13X derivatives of mu-conotoxin GIIIA bind externally to single sodium channels and block current incompletely with mean "blocked" durations of several seconds. We studied interactions between two classes of blockers (mu-conotoxins and amines) by steady state, kinetic analysis of block of BTX-modified Na channels in planar bilayers. The amines cause all-or-none block at a site internal to the selectivity filter. TPrA and DEA block single Na channels with very different kinetics. TPrA induces discrete, all-or-none, blocked events (mean blocked durations, approximately 100 ms), whereas DEA produces a concentration-dependent reduction of the apparent single channel amplitude ("fast" block). These distinct modes of action allow simultaneous evaluation of block by TPrA and DEA, showing a classical, competitive interaction between them. The apparent affinity of TPrA decreases with increasing [DEA], based on a decrease in the association rate for TPrA. When an R13X mu-conotoxin derivative and one of the amines are applied simultaneously on opposite sides of the membrane, a mutually inhibitory interaction is observed. Dissociation constants, at +50 mV, for TPrA ( approximately 4 mM) and DEA ( approximately 30 mM) increase by approximately 20%-50% when R13E (nominal net charge, +4) or R13Q (+5) is bound. Analysis of the slow blocking kinetics for the two toxin derivatives showed comparable decreases in affinity of the mu-conotoxins in the presence of an amine. Although this mutual inhibition seems to be qualitatively consistent with an electrostatic interaction across the selectivity filter, quantitative considerations raise questions about the mechanistic details of the interaction.
- Published
- 2008
4. Residues in Na+ Channel D3-S6 Segment Modulate both Batrachotoxin and Local Anesthetic Affinities
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Carla Nau, Ging Kuo Wang, and Sho-Ya Wang
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Models, Molecular ,Patch-Clamp Techniques ,Mutant ,Molecular Sequence Data ,Biophysics ,Sequence alignment ,Gating ,Transfection ,complex mixtures ,Protein Structure, Secondary ,Sodium Channels ,Cell Line ,Membrane Potentials ,chemistry.chemical_compound ,Animals ,Humans ,Patch clamp ,Amino Acid Sequence ,Anesthetics, Local ,Batrachotoxins ,Muscle, Skeletal ,Membrane potential ,Sodium channel ,Wild type ,Rats ,chemistry ,Biochemistry ,Amino Acid Substitution ,Mutagenesis, Site-Directed ,Batrachotoxin ,Ion Channel Gating ,Sequence Alignment ,Research Article - Abstract
Batrachotoxin (BTX) alters the gating of voltage-gated Na(+) channels and causes these channels to open persistently, whereas local anesthetics (LAs) block Na(+) conductance. The BTX and LA receptors have been mapped to several common residues in D1-S6 and D4-S6 segments of the Na(+) channel alpha-subunit. We substituted individual residues with lysine in homologous segment D3-S6 of the rat muscle mu1 Na(+) channel from F1274 to N1281 to determine whether additional residues are involved in BTX and LA binding. Two mutant channels, mu1-S1276K and mu1-L1280K, when expressed in mammalian cells, become completely resistant to 5 microM BTX during repetitive pulses. The activation and/or fast inactivation gating of these mutants is substantially different from that of wild type. These mutants also display approximately 10-20-fold reduction in bupivacaine affinity toward their inactivated state but show only approximately twofold affinity changes toward their resting state. These results demonstrate that residues mu1-S1276 and mu1-L1280 in D3-S6 are critical for both BTX and LA binding interactions. We propose that LAs interact readily with these residues from D3-S6 along with those from D1-S6 and D4-S6 in close proximity when the Na(+) channel is in its inactivated state. Implications of this state-dependent binding model for the S6 alignment are discussed.
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- 2000
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5. Trimethyloxonium modification of batrachotoxin-activated Na channels alters functionally important protein residues
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D.B. Cherbavaz
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Analytical chemistry ,Biophysics ,Gating ,In Vitro Techniques ,Peptides, Cyclic ,Biophysical Phenomena ,Sodium Channels ,Ion ,chemistry.chemical_compound ,Onium Compounds ,Animals ,Batrachotoxins ,Muscle, Skeletal ,Ion channel ,Chemistry ,Sodium channel ,Electric Conductivity ,Onium compound ,Rats ,Membrane ,Batrachotoxin ,Conotoxins ,Selectivity ,Ion Channel Gating ,Research Article - Abstract
The extracellular side of single batrachotoxin-activated voltage-dependent Na channels isolated from rat skeletal muscle membranes incorporated into neutral planar lipid bilayers were treated in situ with the carboxyl methylating reagent, trimethyloxonium (TMO). These experiments were designed to determine whether TMO alters Na channel function by a general through-space electrostatic mechanism or by methylating specific carboxyl groups essential to channel function. TMO modification reduced single-channel conductance by decreasing the maximal turnover rate. Modification increased channel selectivity for sodium ions relative to potassium ions as measured under biionic conditions. TMO modification increased the mu-conotoxin (muCTX) off-rate by three orders of magnitude. Modification did not alter the muCTX on-rate at low ionic strength or Na channel voltage-dependent gating characteristics. These data demonstrate that TMO does not act via a general electrostatic mechanism. Instead, TMO targets protein residues specifically involved in ion conduction, ion selectivity, and muCTX binding. These data support the hypothesis that muCTX blocks open-channel current by physically obstructing the ion channel pore.
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- 1995
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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.
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- 2016
7. Activation Dynamics of Sodium Ion Channel
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Vijay S. Pande and Matthew P. Harrigan
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medicine.medical_specialty ,Conformational change ,Chemistry ,Sodium channel ,Dynamics (mechanics) ,Biophysics ,Cardiac arrhythmia ,chemistry.chemical_compound ,Endocrinology ,Internal medicine ,Neuropathic pain ,medicine ,Batrachotoxin - Abstract
Voltage gated sodium channels initiate signaling in neurons and other cells which go on to become sensations of pleasure and pain, as well as thoughts and feelings. Sodium channel malfunction has been linked with cardiac arrhythmia, epilepsy, and neuropathic pain. We performed large-scale molecular dynamics simulations of the prokaryotic voltage gated sodium channel to probe dynamics and conformational change of channel activation. In collaboration with experimentalists, we propose a model for binding of batrachotoxin.
- Published
- 2016
8. Transcainide causes two modes of open-channel block with different voltage sensitivities in batrachotoxin-activated sodium channels
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Gerald W. Zamponi and Robert J. French
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Stereochemistry ,Sodium ,Biophysics ,chemistry.chemical_element ,In Vitro Techniques ,Biophysical Phenomena ,Sodium Channels ,Membrane Potentials ,Ion ,chemistry.chemical_compound ,Block (telecommunications) ,Electrochemistry ,Animals ,Batrachotoxins ,Muscle, Skeletal ,Chemistry ,Blocking (radio) ,Myocardium ,Sodium channel ,Lidocaine ,Depolarization ,Rats ,Kinetics ,Membrane ,Verapamil ,Cattle ,Batrachotoxin ,Anti-Arrhythmia Agents ,Sodium Channel Blockers ,Research Article - Abstract
Transcainide, a complex derivative of lidocaine, blocks the open state of BTX-activated sodium channels from bovine heart and rat skeletal muscle in two distinct ways. When applied to either side of the membrane, transcainide caused discrete blocking events a few hundred milliseconds in duration (slow block), and a concomitant reduction in apparent single-channel amplitude, presumably because of rapid block beyond the temporal resolution of our recordings (fast block). We quantitatively analyzed block from the cytoplasmic side. Both modes of block occurred via binding of the drug to the open channel, approximately followed 1:1 stoichiometry, and were similar for both channel subtypes. For slow block, the blocking rate increased, and the unblocking rate decreased with depolarization, yielding an overall enhancement of block at positive potentials, and suggesting a blocking site at an apparent electrical distance about 45% of the way from the cytoplasmic end of the channel (z delta approximately 0.45). In contrast, the fast blocking mode was only slightly enhanced by depolarization (z delta approximately 0.15). Phenomenologically, the bulky and complex transcainide molecule combines the almost voltage-insensitive blocking action of phenylhydrazine (Zamponi and French, 1994a (companion paper)) with a slow open-channel blocking action that shows a voltage dependence typical of simpler amines. Only the slower blocking mode was sensitive to the removal of external sodium ions, suggesting that the two types of block occur at distinct sites. Dose-response relations were also consistent with independent binding of transcainide to two separate sites on the channel.
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- 1994
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9. Fast lidocaine block of cardiac and skeletal muscle sodium channels: one site with two routes of access
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D.D. Doyle, Gerald W. Zamponi, and Robert J. French
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Lidocaine ,Sodium ,Biophysics ,chemistry.chemical_element ,In Vitro Techniques ,Binding, Competitive ,Biophysical Phenomena ,Sodium Channels ,chemistry.chemical_compound ,Electrochemistry ,medicine ,Animals ,Batrachotoxins ,Binding Sites ,Sheep ,Chemistry ,Muscles ,Myocardium ,Sodium channel ,Skeletal muscle ,Heart ,Depolarization ,Anatomy ,Hyperpolarization (biology) ,Rats ,Kinetics ,medicine.anatomical_structure ,Cattle ,Batrachotoxin ,Intracellular ,Research Article ,medicine.drug - Abstract
We have studied the block by lidocaine and its quaternary derivative, QX-314, of single, batrachotoxin (BTX)-activated cardiac and skeletal muscle sodium channels incorporated into planar lipid bilayers. Lidocaine and QX-314, applied to the intracellular side, appear to induce incompletely resolved, rapid transitions between the open and the blocked state of BTX-activated sodium channels from both heart and skeletal muscle. We used amplitude distribution analysis (Yellen, G. 1984. J. Gen. Physiol. 84:157-186.) to estimate the rate constants for block and unblock. Block by lidocaine and QX-314 from the cytoplasmic side exhibits rate constants with similar voltage dependence. The blocking rate increases with depolarization, and the unblocking rate increases with hyperpolarization. Fast lidocaine block was virtually identical for sodium channels from skeletal (rat, sheep) and cardiac (beef, sheep) muscle. Lidocaine block from the extracellular side occurred at similar concentrations. However, for externally applied lidocaine, the blocking rate was voltage-independent, and was proportional to concentration of the uncharged, rather than the charged, form of the drug. In contrast, unblocking rates for internally and externally applied lidocaine were identical in magnitude and voltage dependence. Our kinetic data suggest that lidocaine, coming from the acqueous phase on the cytoplasmic side in the charged form, associates and dissociates freely with the fast block effector site, whereas external lidocaine, in the uncharged form, approaches the same site via a direct, hydrophobic path.
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- 1993
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10. Competitive binding interaction between Zn2+ and saxitoxin in cardiac Na+ channels. Evidence for a sulfhydryl group in the Zn2+/saxitoxin binding site
- Author
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Edward Moczydlowski and L. Schild
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inorganic chemicals ,Stereochemistry ,Sodium ,Molecular Sequence Data ,Biophysics ,chemistry.chemical_element ,In Vitro Techniques ,Binding, Competitive ,Biophysical Phenomena ,Sodium Channels ,Iodoacetamide ,chemistry.chemical_compound ,Dogs ,Animals ,Amino Acid Sequence ,Binding site ,Saxitoxin ,Chemistry ,Sodium channel ,Myocardium ,Dissociation constant ,Kinetics ,Zinc ,Batrachotoxin ,Cattle ,Cysteine ,Research Article - Abstract
Mammalian heart Na+ channels exhibit approximately 100-fold higher affinity for block by external Zn2+ than other Na+ channel subtypes. With batrachotoxin-modified Na+ channels from dog or calf heart, micromolar concentrations of external Zn2+ result in a flickering block to a substate level with a conductance of approximately 12% of the open channel at -50 mV. We examined the hypothesis that, in this blocking mode, Zn2+ binds to a subsite of the saxitoxin (STX) binding site of heart Na+ channels by single-channel analysis of the interaction between Zn2+ and STX and also by chemical modification experiments on single heart Na+ channels incorporated into planar lipid bilayers in the presence of batrachotoxin. We found that external Zn2+ relieved block by STX in a strictly competitive fashion. Kinetic analysis of this phenomenon was consistent with a scheme involving direct binding competition between Zn2+ and STX at a single site with intrinsic equilibrium dissociation constants of 30 nM for STX and 30 microM for Zn2+. Because high-affinity Zn2(+)-binding sites often include sulfhydryl groups as coordinating ligands of this metal ion, we tested the effect of a sulfhydryl-specific alkylating reagent, iodoacetamide (IAA), on Zn2+ and STX block. For six calf heart Na+ channels, we observed that exposure to 5 mM IAA completely abolished Zn2+ block and concomitantly modified STX binding with at least 20-fold reduction in affinity. These results lead us to propose a model in which Zn2+ binds to a subsite within or near the STX binding site of heart Na+ channels. This site is also presumed to contain one or more cysteine sulfhydryl groups.
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- 1991
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11. pH-dependent binding of local anesthetics in single batrachotoxin-activated Na+ channels. Cocaine vs. quaternary compounds
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J. Nettleton and Ging Kuo Wang
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Stereochemistry ,Sodium ,Lipid Bilayers ,education ,Kinetics ,Biophysics ,Synthetic membrane ,chemistry.chemical_element ,Naphthalenes ,Models, Biological ,Sodium Channels ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Cocaine ,030202 anesthesiology ,Animals ,Anesthetics, Local ,Batrachotoxins ,Binding site ,Lipid bilayer ,030304 developmental biology ,0303 health sciences ,Binding Sites ,Chemistry ,Muscles ,Phosphatidylethanolamines ,Lidocaine ,Hydrogen-Ion Concentration ,Pyrrolidinones ,Receptor–ligand kinetics ,3. Good health ,Dissociation constant ,Phosphatidylcholines ,Batrachotoxin ,Rabbits ,Research Article ,Protein Binding - Abstract
The effects of internal and external pH on the binding kinetics of local anesthetics (LAs) were studied in single batrachotoxin-activated Na+ channels incorporated into planar bilayers. With internal quaternary QX-314 and RAC421-II drugs, the binding interactions were little affected by either external or internal pH. With tertiary cocaine, the binding kinetics were drastically altered by pH. A decrease in the internal pH from 9.3 to 6.2 decreased the apparent equilibrium dissociation constant (Kd) of internal cocaine by more than 100-fold. This increase in the binding affinity was mostly accounted for by an increase in the apparent cocaine on-rate constant (kon) of approximately 80-fold. The cocaine off-rate constant (koff) was little changed (between 3–4 s-1). These results demonstrate quantitatively that the charged form of cocaine is the active form for BTX-activated Na+ channels. Surprisingly, the apparent pKa of cocaine near its binding site was estimated to be 1.4 units lower than that in bulk solution (7.1 vs. 8.5), indicating that the LA drug encounters a relatively hydrophobic environment. Opposite to the internal pH effect, a decrease of external pH from 8.4 to 6.2 increased the Kd value of internally and externally applied cocaine by approximately 8- and approximately 25-fold, respectively. External pH effect was primarily mediated by modulation of kon; koff was again relatively unaffected. Our findings support a model in which neutral cocaine can readily cross the membrane barrier, but needs to be protonated internally to bind to its binding site.
- Published
- 1990
12. Charged tetracaine as an inactivation enhancer in batrachotoxin-modified Na+ channels
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Wilson Mok, Shu Wang, and Ging Kuo Wang
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Tetracaine ,Stereochemistry ,Sodium ,Biophysics ,chemistry.chemical_element ,In Vitro Techniques ,Biophysical Phenomena ,Sodium Channels ,Membrane Potentials ,chemistry.chemical_compound ,Sodium channel blocker ,medicine ,Electrochemistry ,Animals ,Binding site ,Batrachotoxins ,Membrane potential ,Binding Sites ,Chemistry ,Sodium channel ,Hydrogen-Ion Concentration ,Clone Cells ,Rats ,Kinetics ,Membrane ,Batrachotoxin ,medicine.drug ,Sodium Channel Blockers ,Research Article - Abstract
Two distinct types of local anesthetics (LAs) have previously been found to block batrachotoxin (BTX)-modified Na+ channels: type 1 LAs such as cocaine and bupivacaine interact preferentially with open channels, whereas type 2 LAs, such as benzocaine and tricaine, with inactivated channels. Herein, we describe our studies of a third type of LA, represented by tetracaine as a dual blocker that binds strongly with closed channels but also binds to a lesser extent with open channels when the membrane is depolarized. Enhanced inactivation of BTX-modified Na+ channels by tetracaine was determined by steady-state inactivation measurement and by the dose-response curve. The 50% inhibitory concentration (IC50) was estimated to be 5.2 microM at -70 mV, where steady-state inactivation was maximal, with a Hill coefficient of 0.98 suggesting that one tetracaine molecule binds with one inactivated channel. Tetracaine also interacted efficiently with Na+ channels when the membrane was depolarized; the IC50 was estimated to be 39.5 microM at +50 mV with a Hill coefficient of 0.94. Unexpectedly, charged tetracaine was found to be the primary active form in the blocking of inactivated channels. In addition, external Na+ ions appeared to antagonize the tetracaine block of inactivated channels. Consistent with these results, N-butyl tetracaine quaternary ammonium, a permanently charged tetracaine derivative, remained a strong inactivation enhancer. Another derivative of tetracaine, 2-(di-methylamino) ethyl benzoate, which lacked a 4-butylamino functional group on the phenyl ring, elicited block that was approximately 100-fold weaker than that of tetracaine. We surmise that 1) the binding site for inactivation enhancers is within the Na+ permeation pathway, 2) external Na+ ions antagonize the block of inactivation enhancers by electrostatic repulsion, 3) the 4-butylamino functional group on the phenyl ring is critical for block and for the enhancement of inactivation, and 4) there are probably overlapping binding sites for both inactivation enhancers and open-channel blockers within the Na+ pore.
- Published
- 1994
13. Ion conduction in substates of the batrachotoxin-modified Na+ channel from toad skeletal muscle
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David Naranjo and Ramon Latorre
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Sodium ,Lipid Bilayers ,Biophysics ,chemistry.chemical_element ,Toad ,In Vitro Techniques ,Sodium Chloride ,Models, Biological ,Biophysical Phenomena ,Sodium Channels ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,biology.animal ,medicine ,Electrochemistry ,Animals ,Protease Inhibitors ,Batrachotoxins ,Lipid bilayer ,030304 developmental biology ,0303 health sciences ,biology ,Sodium channel ,Muscles ,Electric Conductivity ,Conductance ,Skeletal muscle ,Kinetics ,medicine.anatomical_structure ,Membrane ,chemistry ,Biochemistry ,Batrachotoxin ,Anura ,030217 neurology & neurosurgery ,Research Article - Abstract
Batrachotoxin-modified Na+ channels from toad muscle were inserted into planar lipid bilayers composed of neutral phospholipids. Single-channel conductances were measured for [Na+] ranging between 0.4 mM and 3 M. When membrane preparations were made in the absence of protease inhibitors, two open conductance states were identified: a fully open state (16.6 pS in 200 mM symmetrical NaCl) and a substate that was 71% of the full conductance. The substate was predominant at [Na+] > 65 mM, whereas the presence of the fully open state was predominant at [Na+] < 15 mM. Addition of protease inhibitors during membrane preparation stabilized the fully open state over the full range of [Na+] studied. In symmetrical Na+ solutions and in biionic conditions, the ratio of amplitudes remained constant and the two open states exhibited the same permeability ratios of PLi/PNa and PCs/PNa. The current-voltage relations for both states showed inward rectification only at [Na+] < 10 mM, suggesting the presence of asymmetric negative charge densities at both channel entrances, with higher charge density in the external side. An energy barrier profile that includes double ion occupancy and asymmetric charge densities at the channel entrances was required to fit the conductance-[Na+] relations and to account for the rectification seen at low [Na+]. Energy barrier profiles differing only in the energy peaks can give account of the differences between both conductance states. Estimation of the surface charge density at the channel entrances is very dependent on the ion occupancy used and the range of [Na+] tested. Independent evidence for the existence of a charged external vestibule was obtained at low external [Na+] by identical reduction of the outward current induced by micromolar additions of Mg2+ and Ba2+.
- Published
- 1993
14. Is the Skeletal Muscle Sodium Channel of the Batrachotoxin (BTX)-Producing Phyllobates aurotaenia Poison Dart Frog Resistant to BTX?
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Santiago Castano, Ana M. Correa, Walter Sandtner, Ludivine Frezza, Leonardo Fierro, Francisco Bezanilla, and Helberg Asencio
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Phyllobates ,biology ,Toxin ,Sodium channel ,Mutagenesis ,Biophysics ,Xenopus ,Skeletal muscle ,medicine.disease_cause ,biology.organism_classification ,complex mixtures ,chemistry.chemical_compound ,medicine.anatomical_structure ,chemistry ,Biochemistry ,Phyllobates aurotaenia ,medicine ,Batrachotoxin - 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).
- Published
- 2010
15. Modeling ion permeation through batrachotoxin-modified Na+ channels from rat skeletal muscle with a multi-ion pore
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Edward Moczydlowski, O. Alvarez, Arippa Ravindran, H. Kwiecinski, and G. Eisenman
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Lipid Bilayers ,Analytical chemistry ,Biophysics ,In Vitro Techniques ,Lithium ,Mole fraction ,Electrochemistry ,Models, Biological ,Biophysical Phenomena ,Sodium Channels ,Ion ,chemistry.chemical_compound ,Animals ,Surface charge ,Batrachotoxins ,Lipid bilayer ,Muscles ,Sodium ,Conductance ,Permeation ,Rats ,chemistry ,Potassium ,Thermodynamics ,Batrachotoxin ,Research Article - Abstract
The mechanism of ion permeation through Na+ channels that have been modified by batrachotoxin (BTX) and inserted into planar bilayers has been generally described by models based on single-ion occupancy, with or without an influence of negative surface charge, depending on the tissue source. For native Na+ channels there is evidence suggestive of a multi-ion conduction mechanism. To explore the question of ion occupancy, we have reexamined permeation of Na+, Li+, and K+ through BTX-modified Na+ channels from rat skeletal muscle. Single-channel current-voltage (I-V) behavior was studied in neutral lipid bilayers in the presence of symmetrical Na+ concentrations ranging from 0.5 to 3,000 mM. The dependence of unitary current on the mole fraction of Na+ was also examined in symmetrical mixtures of Na(+)-Li+ and Na(+)-K+ at a constant total ionic strength of 206 and 2,006 mM. The dependence of unitary conductance on symmetrical Na+ concentration does not exhibit Michaelis-Menten behavior characteristic of single-ion occupancy but can be simulated by an Eyring-type model with three barriers and two sites (3B2S) that includes double occupancy and ion-ion repulsion. Best-fit energy barrier profiles for Na+, Li+, and K+ were obtained by nonlinear curve fitting of I-V data using the 3B2S model. The Na(+)-Li+ and Na(+)-K+ mole-fraction experiments do not exhibit an anomalous mole-fraction effect. However, the 3B2S model is able to account for the biphasic dependence of unitary conductance on symmetrical [Na+] that is suggestive of multiple occupancy and the monotonic dependence of unitary current on the mole fraction of Na+ that is compatible with single or multiple occupancy. The best-fit 3B2S barrier profiles also successfully predict bi-ionic reversal potentials for Na(+)-Li+ and Na(+)-K+ in both orientations across the channel. Our experimental and modeling results reconcile the dual personality of ion permeation through Na+ channels, which can display features of single or multiple occupancy under various conditions. To a first approximation, the 3B2S model developed for this channel does not require corrections for vestibule surface charge. However, if negative surface charges of the protein do influence conduction, the conductance behavior in the limit of low [Na+] does not correspond to a Gouy-Chapman model of planar surface charge.
- Published
- 1992
16. Interaction between batrachotoxin and yohimbine
- Author
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William A. Catterall, Li-Yen Huang, and Gerald Ehrenstein
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Dose-Response Relationship, Drug ,Stereochemistry ,Sodium channel ,Sodium ,Alkaloid ,Biophysics ,Yohimbine ,chemistry.chemical_element ,Depolarization ,Binding, Competitive ,complex mixtures ,Cell Line ,Dissociation constant ,Neuroblastoma ,chemistry.chemical_compound ,chemistry ,medicine ,Drug Interactions ,Batrachotoxin ,Batrachotoxins ,Veratridine ,Research Article ,medicine.drug - Abstract
The neurotoxins, batrachotoxin and veratridine, are specific activators of sodium channels and cause an increase in the rate of 22Na uptake in neuroblastoma cells. Yohimbine, an indolakylamine alkaloid, inhibits this batrachotoxin-induced 22Na uptake. The dose-response curve of yohimbine suggest that the inhibitor acts reversibly on a single class of binding sites with dissociation constant of 3--4 x 10(-5) M. The dissociation constant is not affected by depolarization from--41 to 0 mV. Kinetic and equilibrium experiments indicate that yohimbine is a competitive inhibitor of the action of batrachotoxin. These results support the conclusion that yohimbine inhibitis the sodium flux by acting on the channel gating mechanism rather than by occluding the channels.
- Published
- 1978
17. Symmetry and asymmetry of permeation through toxin-modified Na+ channels
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Sarah S. Garber
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Cell Membrane Permeability ,Sodium ,Lipid Bilayers ,Analytical chemistry ,Biophysics ,Ionic bonding ,chemistry.chemical_element ,Sodium Channels ,Ion ,Membrane Potentials ,Veratrine ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Animals ,Batrachotoxins ,Lipid bilayer ,030304 developmental biology ,Membrane potential ,0303 health sciences ,Veratridine ,Chemistry ,Sodium channel ,Muscles ,Electric Conductivity ,Cations, Monovalent ,Rats ,Batrachotoxin ,030217 neurology & neurosurgery ,Research Article - Abstract
Single Na+ channels from rat skeletal muscle were inserted into planar lipid bilayers in the presence of either 200 nM batrachotoxin (BTX) or 50 microM veratridine (VT). These toxins, in addition to their ability to shift inactivation of voltage-gated Na+ channels, may be used as probes of ion conduction in these channels. Channels modified by either of the toxins have qualitatively similar selectivity for the alkali cations (Na+ approximately Li+ greater than K+ greater than Rb+ greater than Cs+). Biionic reversal potentials, for example, were concentration independent for all ions studied. Na+/K+ and Na+/Rb+ reversal potentials, however, were dependent on the orientation of the ionic species with respect to the intra- or extracellular face of the channel, whereas Na+/Li+ biionic reversal potentials were not orientation dependent. A simple, four-barrier, three-well, single-ion occupancy model was used to generate current-voltage relationships similar to those observed in symmetrical solutions of Na, K, or Li ions. The barrier profiles for Na and Li ions were symmetric, whereas that for K ions was asymmetric. This suggests the barrier to ion permeation for K ions may be different than that for Na and Li ions. With this model, these hypothetical energy barrier profiles could predict the orientation-dependent reversal potentials observed for Na+/K+ and Na+/Rb+. The energy barrier profiles, however, were not capable of describing biionic Na/Li ion permeation. Together these results support the hypothesis that Na ions have a different rate determining step for ion permeation than that of K and Rb ions.
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- 1988
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18. Batrachotoxin uncouples gating charge immobilization from fast Na inactivation in squid giant axons
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Jay Z. Yeh and J. Tanguy
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Sodium ,Biophysics ,chemistry.chemical_element ,Tetrodotoxin ,Gating ,Pronase ,In Vitro Techniques ,complex mixtures ,Sodium Channels ,Tosyl Compounds ,chemistry.chemical_compound ,Reference Values ,Animals ,Batrachotoxins ,biology ,Chemistry ,Sodium channel ,Chloramines ,Decapodiformes ,Membrane transport ,biology.organism_classification ,Axons ,Kinetics ,Biochemistry ,Batrachotoxin ,Research Article - Abstract
The fast inactivation of sodium currents and the immobolization of sodium gating charge are thought to be closely coupled to each other. This notion was tested in the squid axon in which kinetics and steady-state properties of the gating charge movement were compared before and after removal of the Na inactivation by batrachotoxin (BTX), pronase, or chloramine-T. The immobilization of gating charge was determined by measuring the total charge movement (QON) obtained by integrating the ON gating current (Ig,ON) using a double pulse protocol. After removal of the fast inactivation with pronase or chloramine-T, the gating charge movement was no longer immobilized. In contrast, after BTX modification, the channels still exhibited an immobilization of the gating charge (QON) with an onset time course and voltage dependence similar to that for the activation process. These results show that BTX can uncouple the charge immobilization from the fast Na inactivation mechanism, suggesting that the Na gating charge movement can be immobilized independently of the inactivation of the channel.
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- 1988
19. Fusion of native or reconstituted membranes to liposomes, optimized for single channel recording
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W.S. Agnew and A.M. Correa
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animal structures ,Synthetic membrane ,Biophysics ,Phosphatidylserines ,Models, Biological ,Sodium Channels ,Membrane Potentials ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Animals ,Patch clamp ,Batrachotoxins ,Ion channel ,030304 developmental biology ,0303 health sciences ,Liposome ,Chromatography ,Chemistry ,Phosphatidylethanolamines ,Vesicle ,Cell Membrane ,Biological membrane ,Membrane ,Liposomes ,Phosphatidylcholines ,Batrachotoxin ,030217 neurology & neurosurgery ,Research Article ,Saxitoxin - Abstract
We here describe a protocol for fusing vesicles into large structures suitable for patch clamp recording. The method may be used with native membrane vesicles or with liposomes containing reconstituted/purified ion channels. The resulting unilamellar membranes exhibit high channel surface abundance, yielding multiple channels in the average excised patch. The procedure has been used to record voltage-sensitive Na channels from three native membrane preparations (eel electroplax, rat skeletal muscle, squid optic nerve), and from reconstituted protein purified from eel electroplax. Channels treated with batrachotoxin (BTX) displayed characteristic activation voltage dependence, conductances, selectivity, and sensitivity to saxitoxin (STX).
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- 1988
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20. Influence of negative surface charge on toxin binding to canine heart Na channels in planar bilayers
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Edward Moczydlowski and Arippa Ravindran
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Stereochemistry ,Lipid Bilayers ,Biophysics ,Tetrodotoxin ,Sodium Chloride ,Dissociation (chemistry) ,Sodium Channels ,Divalent ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Dogs ,Animals ,Surface charge ,Batrachotoxins ,Lipid bilayer ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Sodium channel ,Brain ,Heart ,Dissociation constant ,Kinetics ,chemistry ,Batrachotoxin ,030217 neurology & neurosurgery ,Mathematics ,Saxitoxin ,Research Article - Abstract
The presence of negative surface charge near the tetrodotoxin/saxitoxin binding site of canine heart Na channels was revealed by analysis of the kinetics of toxin block of single batrachotoxin-activated Na channels in planar bilayers as a function of [NaCl]. The voltage-dependence of toxin binding and the toxin dissociation rate are nearly constant as [NaCl] is varied from 0.05 to 3 M. In contrast, the association rate constant of the toxins is inversely dependent on [NaCl], with the rate for the divalent toxin, saxitoxin2+, affected more steeply than that of the monovalent toxin, tetrodotoxin1+. These results for toxin-insensitive Na channels from canine heart parallel previous findings for toxin-sensitive Na channels from canine brain. The model of Green et al. (Green, W. N., L. B. Weiss, and O. S. Anderson. 1987. J. Gen. Physiol. 89:873–903), which includes Na+ competition and Gouy-Chapman screening of surface charge, provided an excellent fit to the data. The results suggest that the two canine Na channel subtypes have a similar density of negative surface charge (1 e-/400 A2) and a similar dissociation constant for Na+ competition (0.5 M) at the toxin binding site. Thus, negative surface charge is a conserved feature of channel function of these two subtypes. The difference in toxin binding affinities arises from small differences in intrinsic association and dissociation rates.
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- 1989
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21. Effects of benzocaine on the kinetics of normal and batrachotoxin-modified Na channels in frog node of Ranvier
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J.M. Dubois and M.F. Schneider
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Node of Ranvier ,Chemistry ,Benzocaine ,Sodium ,Time constant ,Biophysics ,Rana esculenta ,Gating ,Anatomy ,Ion Channels ,Rana ,chemistry.chemical_compound ,Kinetics ,medicine.anatomical_structure ,Ranvier's Nodes ,medicine ,GRENOUILLE ,Animals ,Batrachotoxin ,Batrachotoxins ,Ion channel ,medicine.drug ,Research Article - Abstract
The effects of benzocaine (0.5–1 mM) on normal Na currents, and on Na current and gating charge movement (Q) of batrachotoxin (BTX)-modified Na channels were analyzed in voltage-clamped frog node of Ranvier. Without BTX treatment the decay of Na current during pulses to between -40 and 0 mV could be decomposed into two exponential components both in the absence and in the presence of benzocaine. Benzocaine did not significantly alter the inactivation time constant of either component, but reduced both their amplitudes. The amplitude of the slow inactivating component was more decreased by benzocaine than the amplitude of the fast one, leading to an apparently faster decline of the overall Na current. After removal of Na inactivation and charge movement immobilization by BTX, benzocaine decreased the amplitude of INa with no change in time course. INa, QON, and QOFF were all reduced by the same factor. The results suggest that the rate of reaction of benzocaine with its receptor is slow compared to the rates of channel activation and inactivation. The differential effects of benzocaine on the two components of Na current inactivation in normal channels can be explained assuming two types of channel with different rates of inactivation and different affinities for the drug.
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- 1986
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22. State-dependent block underlies the tissue specificity of lidocaine action on batrachotoxin-activated cardiac sodium channels
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Gerald W. Zamponi, D.D. Doyle, and Robert J. French
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Lidocaine ,Sodium ,Lipid Bilayers ,Biophysics ,chemistry.chemical_element ,Gating ,In Vitro Techniques ,Models, Biological ,Sodium Channels ,chemistry.chemical_compound ,medicine ,Animals ,Batrachotoxins ,Lipid bilayer ,Binding Sites ,Dose-Response Relationship, Drug ,Chemistry ,Muscles ,Myocardium ,Sodium channel ,Skeletal muscle ,Anatomy ,Kinetics ,medicine.anatomical_structure ,Mechanism of action ,Cattle ,Batrachotoxin ,medicine.symptom ,Ion Channel Gating ,Research Article ,medicine.drug - Abstract
We have identified two kinetically distinct modes of block, by lidocaine, of cardiac sodium channels, activated by batrachotoxin and incorporated into planar lipid bilayers. Here, we analyze the slow blocking mode which appears as a series of nonconducting events that increase in frequency and duration with increasing lidocaine concentrations. This type of block occurred rarely, if at all, for the skeletal muscle sodium channel subtype. Kinetic analysis showed that a linear open-closed-blocked model is sufficient to account for the major features of our data. Slow block occurs from a long closed state that is a distinguishing characteristic of cardiac channels under these conditions. Slow block showed no significant voltage dependence in the range of -60 to -20 mV for which the detailed kinetic analysis was performed, and was not elicited by application of the permanently charged lidocaine derivative QX-314. By contrast, the fast block, described in the companion paper, results from drug binding to the open state, and is similar for cardiac and skeletal muscle sodium channels. Application of trypsin to the cytoplasmic end of the channel eliminates both the spontaneous, long, gating closures and slow block. Thus, the lidocaine-sensitive closed state of batrachotoxin-activated cardiac sodium channels exhibits a protease susceptibility resembling that of the inactivated state of unmodified sodium channels. It is the slow block caused by lidocaine binding to this closed state that underlies the channel-subtype specificity of lidocaine action in our experiments.
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23. Rapid and Slow Voltage-Dependent Conformational Changes in Segment IVS6 of Voltage-Gated Na+ Channels
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Stephen C. Cannon and Vasanth Vedantham
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Protein Conformation ,Stereochemistry ,Xenopus ,Protein subunit ,Biophysics ,Sodium Channels ,Membrane Potentials ,Reaction rate ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,medicine ,Animals ,Cysteine ,Anesthetics, Local ,Batrachotoxins ,Muscle, Skeletal ,030304 developmental biology ,0303 health sciences ,Tetraethylammonium ,Voltage-gated ion channel ,Sodium channel ,Skeletal muscle ,Valine ,Recombinant Proteins ,Rats ,medicine.anatomical_structure ,Amino Acid Substitution ,chemistry ,Mutagenesis, Site-Directed ,Oocytes ,Female ,Batrachotoxin ,Ion Channel Gating ,030217 neurology & neurosurgery ,Research Article - Abstract
Mutations in segment IVS6 of voltage-gated Na + channels affect fast-inactivation, slow-inactivation, local anesthetic action, and batrachotoxin (BTX) action. To detect conformational changes associated with these processes, we substituted a cysteine for a valine at position 1583 in the rat adult skeletal muscle sodium channel α -subunit, and examined the accessibility of the substituted cysteine to modification by 2-aminoethyl methanethiosulfonate (MTS-EA) in excised macropatches. MTS-EA causes an irreversible reduction in the peak current when applied both internally and externally, with a reaction rate that is strongly voltage-dependent. The rate increased when exposures to MTS-EA occurred during brief conditioning pulses to progressively more depolarized voltages, but decreased when exposures occurred at the end of prolonged depolarizations, revealing two conformational changes near site 1583, one coupled to fast inactivation, and one tightly associated with slow inactivation. Tetraethylammonium, a pore blocker, did not affect the reaction rate from either direction, while BTX, a lipophilic activator of sodium channels, completely prevented the modification reaction from occurring from either direction. We conclude that there are two inactivation-associated conformational changes in the vicinity of site 1583, that the reactive site most likely faces away from the pore, and that site 1583 comprises part of the BTX receptor.
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24. Open-channel block by internally applied amines inhibits activation gate closure in batrachotoxin-activated sodium channels
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Robert J. French and Gerald W. Zamponi
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Diethylamines ,Stereochemistry ,Lipid Bilayers ,Phenylpropanolamine ,Biophysics ,Gating ,In Vitro Techniques ,Models, Biological ,Biophysical Phenomena ,Sodium Channels ,Membrane Potentials ,chemistry.chemical_compound ,Sodium channel blocker ,medicine ,Electrochemistry ,Animals ,Amines ,Batrachotoxins ,Lipid bilayer ,Muscle, Skeletal ,Membrane potential ,Chemistry ,Sodium channel ,Myocardium ,Skeletal muscle ,Lidocaine ,Rats ,Kinetics ,medicine.anatomical_structure ,Batrachotoxin ,Cattle ,Ion Channel Gating ,medicine.drug ,Sodium Channel Blockers ,Research Article - Abstract
We have studied the action of several pore-blocking amines on voltage-dependent activation gating of batrachotoxin(BTX)-activated sodium channels, from bovine heart and rat skeletal muscle, incorporated into planar lipid bilayers. Although structurally simpler, the compounds studied show general structural features and channel-inhibiting actions that resemble those of lidocaine. When applied to the cytoplasmic end of the channel, these compounds cause a rapid, voltage-dependent, open-channel block seen as a reduction in apparent single-channel amplitude (companion paper). Internal application of phenylpropanolamine, phenylethylamine, phenylmethylamine, and diethylamine, as well as causing open-channel block, reduces the probability of channel closure, producing a shift of the steady-state activation curve toward more hyperpolarizing potentials. These gating effects were observed for both cardiac and skeletal muscle channels and were not evoked by addition of equimolar N-Methyl-D-Glucamine, suggesting a specific interaction of the blockers with the channel rather than a surface charge effect. Kinetic analysis of phenylpropanolamine action on skeletal muscle channels indicated that phenylpropanolamine reduced the closed probability via two separate mechanisms. First, mean closed durations were slightly abbreviated in its presence. Second, and more important, the frequency of the gating closures was reduced. This action was correlated with the degree, and the voltage dependence, of open-channel block, suggesting that the activation gate cannot close while the pore is occluded by the blocker. Such a mechanism might underlie the previously reported immobilization of gating charge associated with local anesthetic block of unmodified sodium channels.
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25. Dual actions of procainamide on batrachotoxin-activated sodium channels: open channel block and prevention of inactivation
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Xiaoling Sui, P. W. Codding, Robert J. French, and Gerald W. Zamponi
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Models, Molecular ,Stereochemistry ,Lipid Bilayers ,Biophysics ,Procainamide ,Sodium Channels ,Membrane Potentials ,chemistry.chemical_compound ,Sodium channel blocker ,medicine ,Animals ,Batrachotoxins ,Membrane potential ,Molecular Structure ,Chemistry ,Sodium channel ,Muscles ,Cell Membrane ,Skeletal muscle ,Lidocaine ,Depolarization ,Heart ,Rats ,medicine.anatomical_structure ,Mechanism of action ,Batrachotoxin ,Cattle ,medicine.symptom ,Ion Channel Gating ,medicine.drug ,Sodium Channel Blockers ,Research Article - Abstract
We have investigated the action of procainamide on batrachotoxin (BTX)-activated sodium channels from bovine heart and rat skeletal muscle. When applied to the intracellular side, procainamide induced rapid, open-channel block. We estimated rate constants using amplitude distribution analysis (Yellen, G. 1984. J. Gen. Physiol. 84:157). Membrane depolarization increased the blocking rate and slowed unblock. The rate constants were similar in both magnitude and voltage dependence for cardiac and skeletal muscle channels. Qualitatively, this block resembled the fast open-channel block by lidocaine (Zamponi, G. W., D. D. Doyle, and R. J. French. 1993. Biophys. J. 65:80), but procainamide was about sevenfold less potent. Molecular modeling suggests that the difference in potency between procainamide and lidocaine might arise from the relative orientation of their aromatic rings, or from differences in the structure of the aryl-amine link. For the cardiac channels, procainamide reduced the frequency of transitions to a long-lived closed state which shows features characteristic of inactivation (Zamponi, G. W., D. D. Doyle, and R. J. French. 1993. Biophys J. 65:91). Mean durations of kinetically identified closed states were not affected. The degree of fast block and of inhibition of the slow closures were correlated. Internally applied QX-314, a lidocaine derivative and also a fast blocker, produced a similar effect. Thus, drug binding to the fast blocking site appears to inhibit inactivation in BTX-activated cardiac channels.
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26. Batrachotoxin-Resistant Na+ Channels Derived from Point Mutations in Transmembrane Segment D4-S6
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Sho-Ya Wang and Ging Kuo Wang
- Subjects
Biophysics ,Drug Resistance ,In Vitro Techniques ,Transfection ,Models, Biological ,complex mixtures ,Biophysical Phenomena ,Sodium Channels ,Cell Line ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Cocaine ,medicine ,Animals ,Humans ,Point Mutation ,Binding site ,Anesthetics, Local ,Batrachotoxins ,Receptor ,030304 developmental biology ,0303 health sciences ,Binding Sites ,Chemistry ,Point mutation ,Lysine ,Mutagenesis ,Skeletal muscle ,Depolarization ,Recombinant Proteins ,Rats ,Transmembrane domain ,medicine.anatomical_structure ,Phenotype ,Biochemistry ,Mutagenesis, Site-Directed ,Batrachotoxin ,030217 neurology & neurosurgery ,Research Article - Abstract
Local anesthetics (LAs) block voltage-gated Na+ channels in excitable cells, whereas batrachotoxin (BTX) keeps these channels open persistently. Previous work delimited the LA receptor within the D4-S6 segment of the Na+ channel alpha-subunit, whereas the putative BTX receptor was found within the D1-S6. We mutated residues at D4-S6 critical for LA binding to determine whether such mutations modulate the BTX phenotype in rat skeletal muscle Na+ channels (mu1/rSkm1). We show that mu1-F1579K and mu1-N1584K channels become completely resistant to 5 microM BTX. In contrast, mu1-Y1586K channels remain BTX-sensitive; their fast and slow inactivation is eliminated by BTX after repetitive depolarization. Furthermore, we demonstrate that cocaine elicits a profound time-dependent block after channel activation, consistent with preferential LA binding to BTX-modified open channels. We propose that channel opening promotes better exposure of receptor sites for binding with BTX and LAs, possibly by widening the bordering area around D1-S6, D4-S6, and the pore region. The BTX receptor is probably located at the interface of D1-S6 and D4-S6 segments adjacent to the LA receptor. These two S6 segments may appose too closely to bind BTX and LAs simultaneously when the channel is in its resting closed state.
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27. Permeation of Na+ through open and Zn(2+)-occupied conductance states of cardiac sodium channels modified by batrachotoxin: exploring ion-ion interactions in a multi-ion channel
- Author
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Edward Moczydlowski and Laurent Schild
- Subjects
inorganic chemicals ,Cation binding ,Sodium ,Lipid Bilayers ,Molecular Sequence Data ,Analytical chemistry ,Biophysics ,chemistry.chemical_element ,In Vitro Techniques ,Binding, Competitive ,Models, Biological ,Biophysical Phenomena ,Permeability ,Sodium Channels ,chemistry.chemical_compound ,Dogs ,Animals ,Amino Acid Sequence ,Batrachotoxins ,Lipid bilayer ,Ion channel ,Binding Sites ,Voltage-dependent calcium channel ,Sequence Homology, Amino Acid ,Chemistry ,Sodium channel ,Myocardium ,Electric Conductivity ,Conductance ,Crystallography ,Kinetics ,Zinc ,Thermodynamics ,Batrachotoxin ,Cattle ,Calcium Channels ,Research Article - Abstract
Mammalian heart sodium channels inserted into planar bilayers exhibit a distinctive subconductance state when single batrachotoxin-modified channels are exposed to external Zn2+. The current-voltage behavior of the open state and the Zn(2+)-induced substate was characterized in the presence of symmetrical Na+ ranging from 2 to 3000 mM. The unitary conductance of the open state follows a biphasic dependence on [Na+] that can be accounted for by a 3-barrier-2-site model of Na+ permeation that includes double occupancy and Na(+)-Na+ repulsion. The unitary conductance of the Zn2+ substate follows a monophasic dependence on [Na+] that can be explained by a similar 3-barrier-2-site model with low affinity for Na+ and single occupancy due to repulsive interaction with a Zn2+ ion bound near the external entrance to the pore. The apparent association rate of Zn2+ derived from dwell-time analysis of flickering events is strongly reduced as [Na+] is raised from 50 to 500 mM. The apparent dissociation rate of Zn2+ is also enhanced as [Na+] is increased. While not excluding surface charge effects, such behavior is consistent with two types of ion-ion interactions: 1) A competitive binding interaction between Zn2+ and Na+ due to mutual competition for high affinity sites in close proximity. 2) A noncompetitive, destabilizing interaction resulting from simultaneous occupancy by Zn2+ and Na+. The repulsive influence of Zn2+ on Na+ binding in the cardiac Na+ channel is similar to that which has been proposed to occur between Ca2+ and Na+ in structurally related calcium channels. Based on recent mutagenesis data, a schematic model of functionally important residues in the external cation binding sites of calcium channels and cardiac sodium channels is proposed. In this model, the Zn(2+)-induced subconductance state results from Zn2+ binding to a site in the external vestibule that is close to the entrance of the pore but does not occlude it.
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28. Saxitoxin blocks batrachotoxin-modified sodium channels in the node of Ranvier in a voltage-dependent manner
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Gary R. Strichartz and Thomas A. Rando
- Subjects
Biophysics ,Models, Biological ,Ion Channels ,Rana ,Membrane Potentials ,chemistry.chemical_compound ,Ranvier's Nodes ,medicine ,Neurotoxin ,Animals ,Batrachotoxins ,Membrane potential ,Node of Ranvier ,Chemistry ,Sodium channel ,Rana pipiens ,Sodium ,Anatomy ,Kinetics ,medicine.anatomical_structure ,GRENOUILLE ,Membrane channel ,Batrachotoxin ,Mathematics ,Saxitoxin ,Research Article - Abstract
The inhibition by saxitoxin (STX) of single Na channels incorporated into planar lipid bilayers and modified by batrachotoxin (BTX) previously has been shown to be voltage dependent (Krueger, B.K.,J.F. Worley, and R. J. French, 1983, Nature [Lond.], 303:172–175; Moczydlowski, E., S. Hall, S. S. Garber, G. S. Strichartz, and C. Miller, 1984, J. Gen. Physiol., 84:687–704). We tested for such a voltage dependence of STX block of the Na current in voltage-clamped frog nodes of Ranvier. The block by STX of normal Na channels showed no modulation in response to maintained (20 s) changes of the membrane potential or to a train of brief pulses to potentials more positive than the holding potential. However, when the nodal channels were modified by BTX, the train of pulses produced a modulation of the block of the Na current by STX. The modulation of STX block depended on the voltage of the conditioning pulses and this voltage dependence agreed well with that predicted from the single channel studies over the membrane potential range used in those studies. In addition, we found that the voltage dependence of STX block was manifest only at potentials equal to or more positive than required to activate the channels. Most of the apparent differences among data from single channels in bilayers, equilibrium binding studies of STX, and the experiments described here are resolved by the hypotheses that (a) STX binding to open channels is voltage dependent, and (b) the affinities of STX for closed and inactivated channels are independent of voltage, equal, and less than the open channel affinity at potentials less than 0 mV. Whether these hypotheses apply to the STX block of all Na channels or just of BTX-modified channels remains to be determined.
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29. Evidence for interactions between batrachotoxin-modified channels in hybrid neuroblastoma cells
- Author
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M. Jia, K. Iwasa, Nava Moran, and Gerald Ehrenstein
- Subjects
Sodium channel ,Biophysics ,Hybrid Cells ,Noise (electronics) ,Ion Channels ,Open-channel flow ,Cell Line ,Binomial distribution ,chemistry.chemical_compound ,Kinetics ,Neuroblastoma ,chemistry ,Animals ,Batrachotoxin ,Statistical physics ,Batrachotoxins ,Closing (morphology) ,Ion channel ,Communication channel ,Computer Science::Information Theory ,Research Article - Abstract
Current records from voltage-clamped membrane patches containing two batrachotoxin-modified sodium channels were analyzed to determine whether these channels are identical and independent. In most two-channel patches, the experimentally observed probabilities that zero, one, or two channels are open differ from the binomial distribution, demonstrating that the two channels are nonidentical or nonindependent or both. From the same current records, we also determined the rate for the transition from two open channels to one open channel and for the transition from one open channel to zero open channels. These data are consistent with closing rates for the two channels that are equal and independent. Both probability and closing rate data can be fit by a model wherein the channels are identical, the closing rates are independent, and the opening rate is greater when the other channel is closed than when it is open. The implications of this model for analyzing noise spectra and current variance are examined.
- Published
- 1986
30. Gating kinetics of batrachotoxin-modified sodium channels in neuroblastoma cells determined from single-channel measurements
- Author
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Li-Yen Huang, G. Ehrenstein, and Nava Moran
- Subjects
Sodium ,Voltage clamp ,Models, Neurological ,Biophysics ,Analytical chemistry ,chemistry.chemical_element ,Hybrid Cells ,Ion Channels ,Cell Line ,Membrane Potentials ,chemistry.chemical_compound ,Mice ,Neuroblastoma ,Animals ,Batrachotoxins ,Ion channel ,Membrane potential ,Sodium channel ,Conductance ,Glioma ,Clone Cells ,Rats ,Kinetics ,Membrane ,chemistry ,Batrachotoxin ,Mathematics ,Research Article - Abstract
We have observed the opening and closing of single batrachotoxin (BTX)-modified sodium channels in neuroblastoma cells using the patch-clamp method. The conductance of a single BTX-modified channel is approximately 10 pS. At a given membrane potential, the channels are open longer than are normal sodium channels. As is the case for normal sodium channels, the open dwell times become longer as the membrane is depolarized. For membrane potentials more negative than about -70 mV, histograms of both open-state dwell times and closed-state dwell times could be fit by single exponentials. For more depolarized potentials, although the open-state histograms could still be fit by single exponentials, the closed-state histograms required two exponentials. This data together with macroscopic voltage clamp data on the same system could be accounted for by a three-state closed-closed-open model with transition rates between these states that are exponential functions of membrane potential. One of the implications of this model, in agreement with experiment, is that there are always some closed BTX-modified sodium channels, regardless of membrane potential.
- Published
- 1984
31. Cloning and Sequence Analysis of the Voltage-Gated Muscle Na+ Channel from the Poison Dart Frog Phyllobates aurotaenia
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Francisco Bezanilla, Santiago Castano, Ana M. Correa, Alain J. Labro, Leonardo Fierro, and Ludivine Frezza
- Subjects
Phyllobates ,chemistry.chemical_classification ,biology ,Voltage-gated ion channel ,Sequence analysis ,Biophysics ,Skeletal muscle ,Gating ,biology.organism_classification ,complex mixtures ,Amino acid ,chemistry.chemical_compound ,medicine.anatomical_structure ,chemistry ,Biochemistry ,Phyllobates aurotaenia ,medicine ,Batrachotoxin - 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).
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