Richard Rogart | Yale University (original) (raw)
Papers by Richard Rogart
The Journal of Physiology, 1976
1. A new method of labelling saxitoxin (STX) is described, based on transfer of tritium from trit... more 1. A new method of labelling saxitoxin (STX) is described, based on transfer of tritium from tritiated water to the toxin. 2. The radiochemical purity of the labelled toxin has been directly determined, rather than being based on indirect biochemical means, as in previous experiments with Wilzbach-labelled STX and TTX. 3. The specific activity of the labelled toxin, 66 d.p.m.f-mole-1, corresponds with one tritium atom per molecule STX, an improvement of about 300-fold over other means of labelling TTX and STX. 4. The binding of this toxin to rabbit, lobster and garfish olfactory nerve fibres has been reexamined. 5. The density of sodium channels calculated on the basis of the binding of the toxin is about four to six-times the values previously reported.
The Journal of Physiology, 1977
1. The binding of [3H]saxitoxin to innervated and denervated rat diaphragm muscle, and to normal ... more 1. The binding of [3H]saxitoxin to innervated and denervated rat diaphragm muscle, and to normal frog muscle, has been measured. 2. A saturable component of saxitoxin binding, which was inhibited by tetrodotoxin, was detected in all preparations, as well as a component of non-saturable binding. The values for the maximum saturable capacity, M, and the equilibrium binding constant, K, for normal rat diaphragm muscle were: M = 24-4 f-mole. mg wet-L, and K = 3-8 nM. 3. Denervation of rat diaphragm muscle reduced the maximum binding capacity per unit weight .to 16-5 f-mole. mg-'.tThe value of K remained virtually unchanged at 4-2 nM. 4. It is suggested that the decrease in density per unit weight does not reflect any change in the density of sodium channels per unit area of membrane. 5. Two varieties of the same species of frog, Rana pipiens, were examined. In one variety (Southern) the value of M was 25-6 f-mole. mg-' and the value of K was 4-3 nM. In the Northern variety the maximum binding capacity was less, M being 14-6 f-mole. mg-'; the value of K was 3-8 nM.
Myelin, 1977
Speculating in his Lecons sur l’Histologie du Systeme Nerveux on the physiological function of my... more Speculating in his Lecons sur l’Histologie du Systeme Nerveux on the physiological function of myelin and nodes in conduction in myelinated nerve, Ranvier (1878) concluded that the myelin sheath, because of its lipid makeup, would protect the axon cylinder against compression. While this conclusion does not play a prominent part in current thinking, his second conclusion does—namely, that the myelin might serve as a nonconducting envelope to surround the conducting axon and insulate it electrically from the external conducting medium. Referring to the analogy between conduction in a myelinated nerve and conduction in a marine telegraph cable, Ranvier wrote: But what is the role of the myelin sheath itself? It clearly has a protective role; it preserves the axis-cylinder from compression. As it is liquid, or almost liquid, pressures exerted on it are transmitted in every direction and are thus distributed over many points, so that their constrictive action on the axis-cylinder is much reduced. The myelin has also perhaps another role; it is probably an insulating sheath. One knows that electric wires that are immersed in a conducting medium must be insulated from this medium by a nonconducting sheath; it is on this principle that the construction of submarine cables rests. It would be possible—certain facts lead one to believe—that the transmission of sensory or motor impulses has some analogy with the transmission of electricity, and maybe it is convenient that each nerve tube is insulated so that this transmission is more effective. I do not say, I wish you to note, that this insulating myelin sheath is necessary for the transmission of impulses, since we will see to the contrary, in the next lesson, that this transmission is achieved equally well by nerve fibers that lack myelin; nevertheless, I think that this insulation serves to make it more perfect.
Molecular pharmacology, 1977
Tritium-labeled saxitoxin, which binds saturably to the sodium channels of excitable tissue, also... more Tritium-labeled saxitoxin, which binds saturably to the sodium channels of excitable tissue, also is taken up linearly. Some of the linear uptake seems to represent intracellular uptake of tritiated water. However, the main component of linear uptake is of true toxin and not of radioactive impurity. It results from an accumulation of the divalent toxin in the immediate vicinity of the axonal (and other cell) membranes because of the fixed negative charges on them. A new and general method for determining the radiochemical purity of the exchange-labeled tritiated saxitoxin is described. The temperature dependence of the kinetics of loss of the label from the methylene hydrogens of carbon 12 of labeled saxitoxin is also reported.
The Journal of physiology, 1976
The Journal of physiology, 1976
BY J. M. RITCHIE, R. B. ROGART and G. STRICHARTZ.* Department of Pharmacology, Yale University Sc... more BY J. M. RITCHIE, R. B. ROGART and G. STRICHARTZ.* Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06510, U.S.A. Moore, Narahashi & Shaw (1967) and Keynes, Ritchie & Rojas (1971) used a bio-assay technique to examine the uptake of tetrodotoxin and saxitoxin by lobster and rabbit non-myelinated nerve fibres. A subsequent refinement of the technique (Colquhoun, Henderson & Ritchie, 1972; Henderson, Ritchie & Strichartz, 1973) used tritium-labelled tetrodotoxin ([3H]TTX) and saxitoxin ([3H]STX). However, quantitative accuracy of this latter technique depends on the radiochemical purity of the tritiated toxin, which was estimated indirectly from paper electrophoresis profiles to be more than 85 %. Greater sensitivity in bioassay technique allowed Levinson (1975) to determine more directly the radiochemical purity of [3H]TTX by comparing the binding of toxin to an electric eel electroplax preparation, determined by bio-assay, with the corresponding uptake of [3H]TTX. These experiments indicated that the [3H]TTX used by Levinson (1975) was only about 30% pure. The uptake of toxin by rabbit non-myelinated nerve fibres has, therefore, been re-examined in the light of Levinson's (1975) results. A new method of labelling saxitoxin has been used, utilizing tritium exchange from carrier-free T20 at 50° C, washing in water to remove rapidly exchangeable hydrogen, and final purification by high-voltage paper electrophoresis. The apparent specific radioactivity of the purified toxin was 40'7 c/m-mole (i.e. 1P4 tritium per molecule STX), a substantial improvement over toxin previously labelled by the Wilzbach method which resulted in an apparent 1 tritium per 73 STX, or 285 TTX, molecules (Colquhoun et al. 1972; Henderson et al. 1973). The radiochemical purity of the tritiated saxitoxin, determined by bio-assaying the toxin removed from a soaking solution by rabbit brain or vagi and comparing it with the amount of tritium removed, was about 75 %. Binding to the vagus nerve (at 40 C to prevent back-exchange of tritium) consisted of a small non-saturable linear uptake (0-55 f-mole mg wet-' nM-1) and a hyperbolic saturable component with a maximum uptake of 108 f-mole mg wet-', about four times that determined by Colquhoun et al. (1972) indicating that the latter toxin may also have been quite impure. Analogous experimeIits on frog sartorius muscle similarly showed the
Proceedings of the National Academy of Sciences, 1983
Na+ channels in chick brain and heart have been directly compared by measuring binding of tritium... more Na+ channels in chick brain and heart have been directly compared by measuring binding of tritium-labeled saxitoxin ([3H]STX) to the two tissues under identical conditions. Maximum saturable uptake and toxin affinity were considerably less in chick heart than in chick brain, requiring the development of an assay method to resolve specific [3H]STX uptake in heart. With this method, binding to both preparations consisted of a specific saturable component and a linear nonspecific component. The equilibrium dissociation constant for [3H]STX measured in chick heart (6.2-8.8 nM) was 20-30 times higher than that measured in chick brain (0.3 nM). The dissociation rate for [3H]STX was only about twice as fast in heart as it was in brain, indicating that the decrease in toxin affinity in heart results predominantly from a slowed toxin association rate. The decreased affinity for [3H]STX found at the chick heart Na+ channel is compared with toxin-resistant Na+ channels in other preparations. T...
Proceedings of the National Academy of Sciences, 1977
The density of sodium channels in mammalian myelinated fibers has been estimated from measurement... more The density of sodium channels in mammalian myelinated fibers has been estimated from measurements of the binding of [3H]saxitoxin to rabbit sciatic nerve. Binding both to intact and to homogenized nerve consists of a linear, nonspecific, component and a saturable component that represents binding to the sodium channel. The maximum saturable binding capacity in intact nerve is 19.9 +/- 1.9 fmol-mg wet-1; the equilibrium dissociation constant, Kt, is 3.4 +/- 2.0 nM. Homogenization makes little difference, the maximum binding capacity being 19.9 +/- 1.5 fmol-mg wet-1 with Kt = 1.3 +/- 0.7 nM. These values correspond to a density of about 700,000 sodium channels per node--i.e., about 12,000 per mum2 of nodal membrane. From the difference between the values of maximum saturable binding capacity in intact and homogenized preparation, given the statistical uncertainty of their estimate, it seems that the internodal membrane can have no more than about 25 channels per mum2. The significanc...
The Journal of Membrane Biology, 1979
The binding of saxitoxin, a specific inhibitor of the sodium conductance in excitable membranes, ... more The binding of saxitoxin, a specific inhibitor of the sodium conductance in excitable membranes, has been measured in giant axons from the squid, Loligo pealei. Binding was studied by labeling saxitoxin with tritium, using a solvent-exchange technique, and measuring the toxin uptake by liquid scintillation counting. Total toxin binding is the sum of a saturable, hyperbolic binding component, with a dissociation constant at 2--4 degrees C of 4.3 +/- 1.7 nM (mean SE), and a linear, nonsaturable component. The density of saturable binding sites is 166 +/- 20.4 micrometers-2. From this density and published values of the maximum sodium conductance, the conductance per toxin site is estimated to be about 7 pS, assuming sequential activation and inactivation processes (F. Bezanilla & C.M. Armstrong, 1977, J. Gen. Physiol. 70:549). This single site conductance value of 7 pS is in close aggreement with estimates of the conductance of one open sodium channel from measurements of gating currents and of noise on squid giant axons and is consistent with the hypothesis that one saxitoxin molecule binds to one sodium channel.
Gene, 1997
Voltage-gated Na' channels in mammalian heart differ from those in nerve and skeletal muscle. One... more Voltage-gated Na' channels in mammalian heart differ from those in nerve and skeletal muscle. One major difference is that tetrodotoxin (TTX)-resistant cardiac Na' channels are blocked by 1-10 IAM TTX, whereas TTX-sensitive nerve Na+ channels are blocked by nanomolar TTX concentrations. We constructed a cDNA library from 6-day-old rat hearts, where only low-affinity PH]saxitoxin receptors, corresponding to TTX-resistant Na+ channels, were detected. We isolated several overlapping cDNA clones encompassing 7542 nucleotides and encoding the entire a subunit of a cardiacspecific Na+ channel isoform (designated rat heart I) as well as several rat brain I Na+ channel cDNA clones. The derived amino acid sequence of rat heart I was highly homologous to, but distinct from, previous Na+ channel clones. RNase protection studies showed that the corresponding mRNA species is abundant in newborn and adult rat hearts, but not detectable in brain or innervated skeletal muscle. The same mRNA species appears upon denervation of skeletal muscle, likely accounting for expression of new TTX-resistant Na+ channels. Thus, this cardiac-specific Na+ channel clone appears to encode a distinct TTX-resistant isoform and is another member of the mammalian Na+ channel multigene family, found in newborn heart and denervated skeletal muscles.
Bulletin of Mathematical Biology, 1990
This article concludes a series of papers concerned with the flow of electric current through the... more This article concludes a series of papers concerned with the flow of electric current through the surface membrane of a giant nerve fibre (Hodgkin, Huxley & Katz, 1952; Hodgkin & Huxley, 1952 a-c). Its general object is to
Biophysical Journal, 1994
Sodium channels expressed in oocytes exhibited isoform differences in phasic block by saxitoxin (... more Sodium channels expressed in oocytes exhibited isoform differences in phasic block by saxitoxin (STX). Neuronal channels (rat Ila co-expressed with ,B1 subunit, Br2a + p1) had slower kinetics of phasic block for pulse trains than cardiac channels (RHI). After the membrane was repolarized from a single brief depolarizing step, a test pulse at increasing intervals showed first a decrease in current (post-repolarization block) then eventual recovery in the presence of STX. This block/unblock process for Br2a + (1 was 10-fold slower than that for RHI. A model accounting for these results predicts a faster toxin dissociation rate and a slower association rate for the cardiac isoform, and it also predicts a shorter dwell time in a putative high STX affinity conformation for the cardiac isoform. The RHI mutation (Cys374->Phe), which was previously shown to be neuronal-like with respect to high affinity tonic toxin block, was also neuronal-like with respect to the kinetics of post-repolarization block, suggesting that this single amino acid is important for conferring isoform-specific transition rates determining post-repolarization block. Because the same mutation determines both sensitivity for tonic STX block and the kinetics of phasic STX block, the mechanisms accounting for tonic block and phasic block share the same toxin binding site. We conclude that the residue at position 374, in the putative pore-forming region, confers isoform-specific channel kinetics that underlie phasic toxin block.
Science, 1992
The cardiac sodium channel alpha subunit (RHI) is less sensitive to tetrodotoxin (TTX) and saxito... more The cardiac sodium channel alpha subunit (RHI) is less sensitive to tetrodotoxin (TTX) and saxitoxin (STX) and more sensitive to cadmium than brain and skeletal muscle (microliter) isoforms. An RHI mutant, with Tyr substituted for Cys at position 374 (as in microliter) confers three properties of TTX-sensitive channels: (i) greater sensitivity to TTX (730-fold); (ii) lower sensitivity to cadmium (28-fold); and (iii) altered additional block by toxin upon repetitive stimulation. Thus, the primary determinant of high-affinity TTX-STX binding is a critical aromatic residue at position 374, and the interaction may take place possibly through an ionized hydrogen bond. This finding requires revision of the sodium channel pore structure that has been previously suggested by homology with the potassium channel.
![Research paper thumbnail of Identification of two calcium channel receptor sites for [3H]nitrendipine in mammalian cardiac and smooth muscle membrane](https://attachments.academia-assets.com/89593376/thumbnails/1.jpg)
](Proceedings of the National Academy of Sciences, 1986
Various Ca-channel blockers differ in cardiovascular action despite common effects at the Ca chan... more Various Ca-channel blockers differ in cardiovascular action despite common effects at the Ca channel. Many investigators have reported only a single high-affinity receptor for binding of [3H]nitrendipine, a dihydropyridine Ca-channel blocker. Its equilibrium dissociation constant (Kd) does not match the concentration of nitrendipine needed for a physiological effect on the mammalian cardiac Ca channel. The purpose of these studies was to clarify the existing discrepancy between pharmacological properties of nitrendipine receptors and the physiological effects of the dihydropyridine blockers. Of particular importance in this regard was to provide a pharmacological correlate for electrophysiological studies demonstrating multiple voltage-dependent conformational states of the Ca channel, which show differing affinities for the dihydropyridine Ca-channel blockers. By use of an improved ligand binding assay, our studies demonstrate both "high-affinity" and "low-affinity&q...
Journal of Molecular and Cellular Cardiology, 1991
Annals of the New York Academy of Sciences, 1986
Neuron, 1990
The a subunit of a voltage-sensitive sodium channel characteristic of denervated rat skeletal mus... more The a subunit of a voltage-sensitive sodium channel characteristic of denervated rat skeletal muscle was cloned and characterized. The cDNA encodes a 2018 amino SkM2 mRNA but no detectable SkMl message. These findings suggest that SkM2 is a TTX-insensitive sodium channel expressed in both skeletal and cardiac muscle.
Biophysical Journal, 1994
The rat brain la (Brila) Na channel a-subunit and the brain 1 subunit were coexpressed in Xenopus... more The rat brain la (Brila) Na channel a-subunit and the brain 1 subunit were coexpressed in Xenopus oocytes, and peak whole-oocyte Na current (I) was measured at a test potential of-10 mV. Hyperpolarization of the holding potential resulted in an increased affinity of STX and TTX rested-state block of Brila Na channels. The apparent half-block concentration (EDJ) for STX of Brila current decreased with hyperpolarizing holding potentials (Vhm). At Vhw of-100 mV, the ED50 was 2.1 + 0.4 nM, and the affinity increased to a ED5,of 1.2 0.2 nM with Vhm of-140 mV. In the absence of toxin, the peakcurrent amplitude was the same for all potentals negative to-90 mV, deonsstrating that all of the channels were in a closed conformation and maximally available to open in this range of holding potentals. The Woodhull model (1973) was used to describe the increase of the STX ED5 as a funcbon of holding potential. The equivalent elctrcl distance of blck (8) by STX was 0.18 from the exracellular milieu when the valence of STX was fixed to +2. Analysis of the hoding potential dependence of TTX blck yiekled a similar 8 when the valence of TTX was fixed to + 1. We conclude that the guanidinium toxin site is located partially within the bansmembrane elkctric field. Previous site-directed mutagenesis shtdies demonstrated that an isoforn-specific phenylalanine in the BrIla channel is crical for high affinity toxin block. Therefore, we propose that amino acids at positions corresponding to this Phe in the Brila channel, which lie in the outer vestibule of the channel adjacent to the pore entance, are partially in the transmembrane potential drop.
tance of Na+ channel isoforms in cardiac and NB2a neuroblas-
The Journal of General Physiology, 1993
Monovalent and divalent cations competitively displace tetrodotoxin and saxitoxin (STX) from thei... more Monovalent and divalent cations competitively displace tetrodotoxin and saxitoxin (STX) from their binding sites on nerve and skeletal muscle Na channels. Recent studies of cloned cardiac (toxin-resistant) and brain (toxin-sensitive) Na channels suggest important structural differences in their toxin and divalent cation binding sites. We used a partially purified preparation of sheep cardiac Na channels to compare monovalent and divalent cation competition and pH dependence of binding of [3H]STX between these toxin-resistant channels and toxin-sensitive channels in membranes prepared from rat brain. The effects of several chemical modifiers of amino acid groups were also compared. Toxin competition curves for Na+ in heart and Cd2+ in brain yielded similar KD values to measurements of equilibrium binding curves. The monovalent cation sequence for effectiveness of [3H]STX competition is the same for cardiac and brain Na channels, with similar KI values for each ion and slopes of -1. T...
The Journal of Physiology, 1976
1. A new method of labelling saxitoxin (STX) is described, based on transfer of tritium from trit... more 1. A new method of labelling saxitoxin (STX) is described, based on transfer of tritium from tritiated water to the toxin. 2. The radiochemical purity of the labelled toxin has been directly determined, rather than being based on indirect biochemical means, as in previous experiments with Wilzbach-labelled STX and TTX. 3. The specific activity of the labelled toxin, 66 d.p.m.f-mole-1, corresponds with one tritium atom per molecule STX, an improvement of about 300-fold over other means of labelling TTX and STX. 4. The binding of this toxin to rabbit, lobster and garfish olfactory nerve fibres has been reexamined. 5. The density of sodium channels calculated on the basis of the binding of the toxin is about four to six-times the values previously reported.
The Journal of Physiology, 1977
1. The binding of [3H]saxitoxin to innervated and denervated rat diaphragm muscle, and to normal ... more 1. The binding of [3H]saxitoxin to innervated and denervated rat diaphragm muscle, and to normal frog muscle, has been measured. 2. A saturable component of saxitoxin binding, which was inhibited by tetrodotoxin, was detected in all preparations, as well as a component of non-saturable binding. The values for the maximum saturable capacity, M, and the equilibrium binding constant, K, for normal rat diaphragm muscle were: M = 24-4 f-mole. mg wet-L, and K = 3-8 nM. 3. Denervation of rat diaphragm muscle reduced the maximum binding capacity per unit weight .to 16-5 f-mole. mg-'.tThe value of K remained virtually unchanged at 4-2 nM. 4. It is suggested that the decrease in density per unit weight does not reflect any change in the density of sodium channels per unit area of membrane. 5. Two varieties of the same species of frog, Rana pipiens, were examined. In one variety (Southern) the value of M was 25-6 f-mole. mg-' and the value of K was 4-3 nM. In the Northern variety the maximum binding capacity was less, M being 14-6 f-mole. mg-'; the value of K was 3-8 nM.
Myelin, 1977
Speculating in his Lecons sur l’Histologie du Systeme Nerveux on the physiological function of my... more Speculating in his Lecons sur l’Histologie du Systeme Nerveux on the physiological function of myelin and nodes in conduction in myelinated nerve, Ranvier (1878) concluded that the myelin sheath, because of its lipid makeup, would protect the axon cylinder against compression. While this conclusion does not play a prominent part in current thinking, his second conclusion does—namely, that the myelin might serve as a nonconducting envelope to surround the conducting axon and insulate it electrically from the external conducting medium. Referring to the analogy between conduction in a myelinated nerve and conduction in a marine telegraph cable, Ranvier wrote: But what is the role of the myelin sheath itself? It clearly has a protective role; it preserves the axis-cylinder from compression. As it is liquid, or almost liquid, pressures exerted on it are transmitted in every direction and are thus distributed over many points, so that their constrictive action on the axis-cylinder is much reduced. The myelin has also perhaps another role; it is probably an insulating sheath. One knows that electric wires that are immersed in a conducting medium must be insulated from this medium by a nonconducting sheath; it is on this principle that the construction of submarine cables rests. It would be possible—certain facts lead one to believe—that the transmission of sensory or motor impulses has some analogy with the transmission of electricity, and maybe it is convenient that each nerve tube is insulated so that this transmission is more effective. I do not say, I wish you to note, that this insulating myelin sheath is necessary for the transmission of impulses, since we will see to the contrary, in the next lesson, that this transmission is achieved equally well by nerve fibers that lack myelin; nevertheless, I think that this insulation serves to make it more perfect.
Molecular pharmacology, 1977
Tritium-labeled saxitoxin, which binds saturably to the sodium channels of excitable tissue, also... more Tritium-labeled saxitoxin, which binds saturably to the sodium channels of excitable tissue, also is taken up linearly. Some of the linear uptake seems to represent intracellular uptake of tritiated water. However, the main component of linear uptake is of true toxin and not of radioactive impurity. It results from an accumulation of the divalent toxin in the immediate vicinity of the axonal (and other cell) membranes because of the fixed negative charges on them. A new and general method for determining the radiochemical purity of the exchange-labeled tritiated saxitoxin is described. The temperature dependence of the kinetics of loss of the label from the methylene hydrogens of carbon 12 of labeled saxitoxin is also reported.
The Journal of physiology, 1976
The Journal of physiology, 1976
BY J. M. RITCHIE, R. B. ROGART and G. STRICHARTZ.* Department of Pharmacology, Yale University Sc... more BY J. M. RITCHIE, R. B. ROGART and G. STRICHARTZ.* Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06510, U.S.A. Moore, Narahashi & Shaw (1967) and Keynes, Ritchie & Rojas (1971) used a bio-assay technique to examine the uptake of tetrodotoxin and saxitoxin by lobster and rabbit non-myelinated nerve fibres. A subsequent refinement of the technique (Colquhoun, Henderson & Ritchie, 1972; Henderson, Ritchie & Strichartz, 1973) used tritium-labelled tetrodotoxin ([3H]TTX) and saxitoxin ([3H]STX). However, quantitative accuracy of this latter technique depends on the radiochemical purity of the tritiated toxin, which was estimated indirectly from paper electrophoresis profiles to be more than 85 %. Greater sensitivity in bioassay technique allowed Levinson (1975) to determine more directly the radiochemical purity of [3H]TTX by comparing the binding of toxin to an electric eel electroplax preparation, determined by bio-assay, with the corresponding uptake of [3H]TTX. These experiments indicated that the [3H]TTX used by Levinson (1975) was only about 30% pure. The uptake of toxin by rabbit non-myelinated nerve fibres has, therefore, been re-examined in the light of Levinson's (1975) results. A new method of labelling saxitoxin has been used, utilizing tritium exchange from carrier-free T20 at 50° C, washing in water to remove rapidly exchangeable hydrogen, and final purification by high-voltage paper electrophoresis. The apparent specific radioactivity of the purified toxin was 40'7 c/m-mole (i.e. 1P4 tritium per molecule STX), a substantial improvement over toxin previously labelled by the Wilzbach method which resulted in an apparent 1 tritium per 73 STX, or 285 TTX, molecules (Colquhoun et al. 1972; Henderson et al. 1973). The radiochemical purity of the tritiated saxitoxin, determined by bio-assaying the toxin removed from a soaking solution by rabbit brain or vagi and comparing it with the amount of tritium removed, was about 75 %. Binding to the vagus nerve (at 40 C to prevent back-exchange of tritium) consisted of a small non-saturable linear uptake (0-55 f-mole mg wet-' nM-1) and a hyperbolic saturable component with a maximum uptake of 108 f-mole mg wet-', about four times that determined by Colquhoun et al. (1972) indicating that the latter toxin may also have been quite impure. Analogous experimeIits on frog sartorius muscle similarly showed the
Proceedings of the National Academy of Sciences, 1983
Na+ channels in chick brain and heart have been directly compared by measuring binding of tritium... more Na+ channels in chick brain and heart have been directly compared by measuring binding of tritium-labeled saxitoxin ([3H]STX) to the two tissues under identical conditions. Maximum saturable uptake and toxin affinity were considerably less in chick heart than in chick brain, requiring the development of an assay method to resolve specific [3H]STX uptake in heart. With this method, binding to both preparations consisted of a specific saturable component and a linear nonspecific component. The equilibrium dissociation constant for [3H]STX measured in chick heart (6.2-8.8 nM) was 20-30 times higher than that measured in chick brain (0.3 nM). The dissociation rate for [3H]STX was only about twice as fast in heart as it was in brain, indicating that the decrease in toxin affinity in heart results predominantly from a slowed toxin association rate. The decreased affinity for [3H]STX found at the chick heart Na+ channel is compared with toxin-resistant Na+ channels in other preparations. T...
Proceedings of the National Academy of Sciences, 1977
The density of sodium channels in mammalian myelinated fibers has been estimated from measurement... more The density of sodium channels in mammalian myelinated fibers has been estimated from measurements of the binding of [3H]saxitoxin to rabbit sciatic nerve. Binding both to intact and to homogenized nerve consists of a linear, nonspecific, component and a saturable component that represents binding to the sodium channel. The maximum saturable binding capacity in intact nerve is 19.9 +/- 1.9 fmol-mg wet-1; the equilibrium dissociation constant, Kt, is 3.4 +/- 2.0 nM. Homogenization makes little difference, the maximum binding capacity being 19.9 +/- 1.5 fmol-mg wet-1 with Kt = 1.3 +/- 0.7 nM. These values correspond to a density of about 700,000 sodium channels per node--i.e., about 12,000 per mum2 of nodal membrane. From the difference between the values of maximum saturable binding capacity in intact and homogenized preparation, given the statistical uncertainty of their estimate, it seems that the internodal membrane can have no more than about 25 channels per mum2. The significanc...
The Journal of Membrane Biology, 1979
The binding of saxitoxin, a specific inhibitor of the sodium conductance in excitable membranes, ... more The binding of saxitoxin, a specific inhibitor of the sodium conductance in excitable membranes, has been measured in giant axons from the squid, Loligo pealei. Binding was studied by labeling saxitoxin with tritium, using a solvent-exchange technique, and measuring the toxin uptake by liquid scintillation counting. Total toxin binding is the sum of a saturable, hyperbolic binding component, with a dissociation constant at 2--4 degrees C of 4.3 +/- 1.7 nM (mean SE), and a linear, nonsaturable component. The density of saturable binding sites is 166 +/- 20.4 micrometers-2. From this density and published values of the maximum sodium conductance, the conductance per toxin site is estimated to be about 7 pS, assuming sequential activation and inactivation processes (F. Bezanilla & C.M. Armstrong, 1977, J. Gen. Physiol. 70:549). This single site conductance value of 7 pS is in close aggreement with estimates of the conductance of one open sodium channel from measurements of gating currents and of noise on squid giant axons and is consistent with the hypothesis that one saxitoxin molecule binds to one sodium channel.
Gene, 1997
Voltage-gated Na' channels in mammalian heart differ from those in nerve and skeletal muscle. One... more Voltage-gated Na' channels in mammalian heart differ from those in nerve and skeletal muscle. One major difference is that tetrodotoxin (TTX)-resistant cardiac Na' channels are blocked by 1-10 IAM TTX, whereas TTX-sensitive nerve Na+ channels are blocked by nanomolar TTX concentrations. We constructed a cDNA library from 6-day-old rat hearts, where only low-affinity PH]saxitoxin receptors, corresponding to TTX-resistant Na+ channels, were detected. We isolated several overlapping cDNA clones encompassing 7542 nucleotides and encoding the entire a subunit of a cardiacspecific Na+ channel isoform (designated rat heart I) as well as several rat brain I Na+ channel cDNA clones. The derived amino acid sequence of rat heart I was highly homologous to, but distinct from, previous Na+ channel clones. RNase protection studies showed that the corresponding mRNA species is abundant in newborn and adult rat hearts, but not detectable in brain or innervated skeletal muscle. The same mRNA species appears upon denervation of skeletal muscle, likely accounting for expression of new TTX-resistant Na+ channels. Thus, this cardiac-specific Na+ channel clone appears to encode a distinct TTX-resistant isoform and is another member of the mammalian Na+ channel multigene family, found in newborn heart and denervated skeletal muscles.
Bulletin of Mathematical Biology, 1990
This article concludes a series of papers concerned with the flow of electric current through the... more This article concludes a series of papers concerned with the flow of electric current through the surface membrane of a giant nerve fibre (Hodgkin, Huxley & Katz, 1952; Hodgkin & Huxley, 1952 a-c). Its general object is to
Biophysical Journal, 1994
Sodium channels expressed in oocytes exhibited isoform differences in phasic block by saxitoxin (... more Sodium channels expressed in oocytes exhibited isoform differences in phasic block by saxitoxin (STX). Neuronal channels (rat Ila co-expressed with ,B1 subunit, Br2a + p1) had slower kinetics of phasic block for pulse trains than cardiac channels (RHI). After the membrane was repolarized from a single brief depolarizing step, a test pulse at increasing intervals showed first a decrease in current (post-repolarization block) then eventual recovery in the presence of STX. This block/unblock process for Br2a + (1 was 10-fold slower than that for RHI. A model accounting for these results predicts a faster toxin dissociation rate and a slower association rate for the cardiac isoform, and it also predicts a shorter dwell time in a putative high STX affinity conformation for the cardiac isoform. The RHI mutation (Cys374->Phe), which was previously shown to be neuronal-like with respect to high affinity tonic toxin block, was also neuronal-like with respect to the kinetics of post-repolarization block, suggesting that this single amino acid is important for conferring isoform-specific transition rates determining post-repolarization block. Because the same mutation determines both sensitivity for tonic STX block and the kinetics of phasic STX block, the mechanisms accounting for tonic block and phasic block share the same toxin binding site. We conclude that the residue at position 374, in the putative pore-forming region, confers isoform-specific channel kinetics that underlie phasic toxin block.
Science, 1992
The cardiac sodium channel alpha subunit (RHI) is less sensitive to tetrodotoxin (TTX) and saxito... more The cardiac sodium channel alpha subunit (RHI) is less sensitive to tetrodotoxin (TTX) and saxitoxin (STX) and more sensitive to cadmium than brain and skeletal muscle (microliter) isoforms. An RHI mutant, with Tyr substituted for Cys at position 374 (as in microliter) confers three properties of TTX-sensitive channels: (i) greater sensitivity to TTX (730-fold); (ii) lower sensitivity to cadmium (28-fold); and (iii) altered additional block by toxin upon repetitive stimulation. Thus, the primary determinant of high-affinity TTX-STX binding is a critical aromatic residue at position 374, and the interaction may take place possibly through an ionized hydrogen bond. This finding requires revision of the sodium channel pore structure that has been previously suggested by homology with the potassium channel.
Proceedings of the National Academy of Sciences, 1986
Various Ca-channel blockers differ in cardiovascular action despite common effects at the Ca chan... more Various Ca-channel blockers differ in cardiovascular action despite common effects at the Ca channel. Many investigators have reported only a single high-affinity receptor for binding of [3H]nitrendipine, a dihydropyridine Ca-channel blocker. Its equilibrium dissociation constant (Kd) does not match the concentration of nitrendipine needed for a physiological effect on the mammalian cardiac Ca channel. The purpose of these studies was to clarify the existing discrepancy between pharmacological properties of nitrendipine receptors and the physiological effects of the dihydropyridine blockers. Of particular importance in this regard was to provide a pharmacological correlate for electrophysiological studies demonstrating multiple voltage-dependent conformational states of the Ca channel, which show differing affinities for the dihydropyridine Ca-channel blockers. By use of an improved ligand binding assay, our studies demonstrate both "high-affinity" and "low-affinity&q...
Journal of Molecular and Cellular Cardiology, 1991
Annals of the New York Academy of Sciences, 1986
Neuron, 1990
The a subunit of a voltage-sensitive sodium channel characteristic of denervated rat skeletal mus... more The a subunit of a voltage-sensitive sodium channel characteristic of denervated rat skeletal muscle was cloned and characterized. The cDNA encodes a 2018 amino SkM2 mRNA but no detectable SkMl message. These findings suggest that SkM2 is a TTX-insensitive sodium channel expressed in both skeletal and cardiac muscle.
Biophysical Journal, 1994
The rat brain la (Brila) Na channel a-subunit and the brain 1 subunit were coexpressed in Xenopus... more The rat brain la (Brila) Na channel a-subunit and the brain 1 subunit were coexpressed in Xenopus oocytes, and peak whole-oocyte Na current (I) was measured at a test potential of-10 mV. Hyperpolarization of the holding potential resulted in an increased affinity of STX and TTX rested-state block of Brila Na channels. The apparent half-block concentration (EDJ) for STX of Brila current decreased with hyperpolarizing holding potentials (Vhm). At Vhw of-100 mV, the ED50 was 2.1 + 0.4 nM, and the affinity increased to a ED5,of 1.2 0.2 nM with Vhm of-140 mV. In the absence of toxin, the peakcurrent amplitude was the same for all potentals negative to-90 mV, deonsstrating that all of the channels were in a closed conformation and maximally available to open in this range of holding potentals. The Woodhull model (1973) was used to describe the increase of the STX ED5 as a funcbon of holding potential. The equivalent elctrcl distance of blck (8) by STX was 0.18 from the exracellular milieu when the valence of STX was fixed to +2. Analysis of the hoding potential dependence of TTX blck yiekled a similar 8 when the valence of TTX was fixed to + 1. We conclude that the guanidinium toxin site is located partially within the bansmembrane elkctric field. Previous site-directed mutagenesis shtdies demonstrated that an isoforn-specific phenylalanine in the BrIla channel is crical for high affinity toxin block. Therefore, we propose that amino acids at positions corresponding to this Phe in the Brila channel, which lie in the outer vestibule of the channel adjacent to the pore entance, are partially in the transmembrane potential drop.
tance of Na+ channel isoforms in cardiac and NB2a neuroblas-
The Journal of General Physiology, 1993
Monovalent and divalent cations competitively displace tetrodotoxin and saxitoxin (STX) from thei... more Monovalent and divalent cations competitively displace tetrodotoxin and saxitoxin (STX) from their binding sites on nerve and skeletal muscle Na channels. Recent studies of cloned cardiac (toxin-resistant) and brain (toxin-sensitive) Na channels suggest important structural differences in their toxin and divalent cation binding sites. We used a partially purified preparation of sheep cardiac Na channels to compare monovalent and divalent cation competition and pH dependence of binding of [3H]STX between these toxin-resistant channels and toxin-sensitive channels in membranes prepared from rat brain. The effects of several chemical modifiers of amino acid groups were also compared. Toxin competition curves for Na+ in heart and Cd2+ in brain yielded similar KD values to measurements of equilibrium binding curves. The monovalent cation sequence for effectiveness of [3H]STX competition is the same for cardiac and brain Na channels, with similar KI values for each ion and slopes of -1. T...