RESUMO
Unitary conductances of native Na+ channel isoforms (gamma Na) have been determined under a variety of conditions, making comparisons of gamma Na difficult. To allow direct comparison, we measured gamma Na in cell-attached patches on NB2a neuroblastoma cells and rabbit ventricular myocytes under identical conditions [pipette solution (in mM): 280 Na+ and 2 Ca2+, pH 7.4; 10 degrees C]. gamma Na of NB2a channels, 22.9 +/- 0.9 pS, was 21% greater than that of cardiac channels, 18.9 +/- 0.9 pS. In contrast, respective extrapolated reversal potentials, +62.4 +/- 4.6 and +57.9 +/- 5.1 mV, were not significantly different. Several kinetic differences between the channel types were also noted. Negative to -20 mV, mean open time (MOT) of the NB2a isoform was significantly less than that of cardiac channels, and, near threshold, latency to channel opening decayed more rapidly in NB2a. On the basis of analysis of MOT between -60 and 0 mV, the rate constants at 0 mV for the open-to-closed (O-->C) and open-to-inactivated (O-->I) transitions were 0.42 +/- 0.11 and 0.47 +/- 0.11 ms-1 in NB2a and 0.10 +/- 0.06 and 1.19 +/- 0.07 ms-1 in myocytes. The slope factors were -38.9 +/- 8.7 and +10.7 +/- 6.1 mV in NB2a and -27.3 +/- 7.1 and +23.7 +/- 4.9 mV in myocytes. Transition rate constants were significantly different in NB2a and cardiac cells, but voltage dependence was not.
Assuntos
Miocárdio/metabolismo , Neuroblastoma/metabolismo , Canais de Sódio/fisiologia , Animais , Condutividade Elétrica , Ventrículos do Coração , Ativação do Canal Iônico , Isomerismo , Cinética , Camundongos , Modelos Biológicos , Miocárdio/citologia , Neuroblastoma/patologia , Técnicas de Patch-Clamp , Coelhos , Células Tumorais CultivadasRESUMO
The rat brain IIa (BrIIa) Na channel alpha-subunit and the brain beta 1 subunit were coexpressed in Xenopus oocytes, and peak whole-oocyte Na current (INa) 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 BrIIa Na channels. The apparent half-block concentration (ED50) for STX of BrIIa current decreased with hyperpolarizing holding potentials (Vhold). At Vhold of -100 mV, the ED50 was 2.1 +/- 0.4 nM, and the affinity increased to a ED50 of 1.2 +/- 0.2 nM with Vhold of -140 mV. In the absence of toxin, the peak current amplitude was the same for all potentials negative to -90 mV, demonstrating that all of the channels were in a closed conformation and maximally available to open in this range of holding potentials. The Woodhull model (1973) was used to describe the increase of the STX ED50 as a function of holding potential. The equivalent electrical distance of block (delta) by STX was 0.18 from the extracellular milieu when the valence of STX was fixed to +2. Analysis of the holding potential dependence of TTX block yielded a similar delta when the valence of TTX was fixed to +1. We conclude that the guanidinium toxin site is located partially within the transmembrane electric field. Previous site-directed mutagenesis studies demonstrated that an isoform-specific phenylalanine in the BrIIa channel is critical for high affinity toxin block. Therefore, we propose that amino acids at positions corresponding to this Phe in the BrIIa channel, which lie in the outer vestibule of the channel adjacent to the pore entrance,are partially in the transmembrane potential drop.
Assuntos
Saxitoxina/metabolismo , Canais de Sódio/metabolismo , Tetrodotoxina/metabolismo , Animais , Sítios de Ligação , Fenômenos Biofísicos , Biofísica , Encéfalo/metabolismo , Clonagem Molecular , Eletroquímica , Feminino , Técnicas In Vitro , Potenciais da Membrana , Estrutura Molecular , Oócitos/metabolismo , Ratos , Canais de Sódio/química , Canais de Sódio/genética , XenopusRESUMO
Sodium channels expressed in oocytes exhibited isoform differences in phasic block by saxitoxin (STX). Neuronal channels (rat IIa co-expressed with beta 1 subunit, Br2a + beta 1) 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 + beta 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.
Assuntos
Saxitoxina/toxicidade , Bloqueadores dos Canais de Sódio , Animais , Fenômenos Biofísicos , Biofísica , Clonagem Molecular , Resistência a Medicamentos , Feminino , Técnicas In Vitro , Cinética , Potenciais da Membrana/efeitos dos fármacos , Modelos Biológicos , Mutagênese Sítio-Dirigida , Miocárdio/metabolismo , Neurônios/metabolismo , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Canais de Sódio/genética , Canais de Sódio/metabolismo , XenopusRESUMO
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. The effectiveness sequence corresponds to unhydrated ion radii. For seven divalent cations tested (Ca2+, Mg2+, Mn2+, Co2+, Ni2+, Cd2+, and Zn2+) the sequence for [3H]STX competition was also similar. However, whereas all ions displaced [3H]STX from cardiac Na channels at lower concentrations, Cd2+ and Zn2+ did so at much lower concentrations. In addition, and by way of explication, the divalent ion competition curves for both brain and cardiac channels (except for Cd2+ and Zn2+ in heart and Zn2+ in brain) had slopes of less than -1, consistent with more than one interaction site. Two-site curves had statistically better fits than one-site curves. The derived values of KI for the higher affinity sites were similar between the channel types, but the lower affinity KI's were larger for heart. On the other hand, the slopes of competition curves for Cd2+ and Zn2+ were close to -1, as if the cardiac Na channel had one dominant site of interaction or more than one site with similar values for KI. pH titration of [3H]STX binding to cardiac channels showed a pKa of 5.5 and a slope of 0.6-0.9, compared with a pKa of 5.1 and slope of 1 for brain channels. Tetramethyloxonium (TMO) treatment abolished [3H]STX binding to cardiac and brain channels and STX protected channels, but the TMO effect was less dramatic for cardiac channels. Trinitrobenzene sulfonate preferentially abolished [3H]STX binding to brain channels by action at an STX protected site.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Cátions Bivalentes/metabolismo , Saxitoxina/metabolismo , Canais de Sódio/metabolismo , Tetrodotoxina/farmacologia , Animais , Sítios de Ligação/efeitos dos fármacos , Ligação Competitiva/efeitos dos fármacos , Química Encefálica/efeitos dos fármacos , Química Encefálica/fisiologia , Cátions Bivalentes/farmacologia , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Cinética , Modelos Biológicos , Miocárdio/metabolismo , Ratos , Ratos Sprague-Dawley , Saxitoxina/química , Ovinos , Canais de Sódio/química , Canais de Sódio/efeitos dos fármacosRESUMO
The neonatal rat cardiac Na channel alpha-subunit directed currents in oocytes show characteristic cardiac relative resistance to tetrodotoxin (TTX) block. TTX-sensitive currents obtained by expression in Xenopus oocytes of the alpha-subunits of the rat brain (BrnIIa) and adult skeletal muscle (microI) Na channels show abnormally slow decay kinetics. In order to determine if currents directed by the cardiac alpha-subunit (RHI) exhibit kinetics in oocytes like native currents, we compared RHI-directed currents in oocytes to Na currents in freshly isolated neonatal rat myocytes. The decay rate of RHI currents approached that of neonatal myocytes and was faster than BrnIIa and microI currents in oocytes. The voltage dependence of availability and activation was the same as that in the rat myocytes except for a 12-19 mV shift in the depolarizing direction. The RHI Na currents were sensitive to Cd2+ block, and they showed use dependence of TTX and lidocaine block similar to native currents. The current expressed in oocytes following injection of the cRNA encoding for the alpha-subunit of the cardiac Na channel possesses most of the characteristic kinetic and pharmacological properties of the native cardiac Na current.
Assuntos
Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Oócitos/fisiologia , Canais de Sódio/efeitos dos fármacos , Canais de Sódio/genética , Animais , Clonagem Molecular , Feminino , Expressão Gênica/efeitos dos fármacos , Coração/fisiologia , Lidocaína/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Oócitos/química , Ratos , Canais de Sódio/química , Tetrodotoxina/farmacologia , Xenopus laevisRESUMO
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.
Assuntos
Coração/fisiologia , Mutagênese Sítio-Dirigida , Oócitos/fisiologia , Canais de Sódio/fisiologia , Tetrodotoxina/farmacologia , Sequência de Aminoácidos , Animais , Encéfalo/fisiologia , Cádmio/farmacologia , Membrana Celular/fisiologia , Clonagem Molecular , Resistência a Medicamentos/genética , Vetores Genéticos , Cinética , Dados de Sequência Molecular , Músculos/fisiologia , Oócitos/efeitos dos fármacos , Reação em Cadeia da Polimerase , Conformação Proteica , RNA/genética , Ratos , Mapeamento por Restrição , Saxitoxina/farmacologia , Canais de Sódio/efeitos dos fármacos , Canais de Sódio/genética , XenopusRESUMO
We describe the expression of functional Na+ channels in Xenopus oocytes injected with cRNA transcribed from the rat heart I cDNA clone. The expressed rat heart I Na+ currents show kinetic properties and resistance to tetrodotoxin and saxitoxin which are characteristic of native cardiac Na+ currents. The primary amino acid sequence of the rat heart I alpha-subunit is therefore sufficient for expression of tetrodotoxin resistance, and the rat heart I clone is likely to account for the tetrodotoxin-resistant phenotype of cardiac and denervated skeletal muscle.
Assuntos
Canais de Sódio/genética , Animais , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Eletrofisiologia , Expressão Gênica , Microinjeções , Dados de Sequência Molecular , Miocárdio , Oligonucleotídeos , RNA Mensageiro/genética , Saxitoxina/metabolismo , Saxitoxina/farmacologia , Sódio/fisiologia , Canais de Sódio/efeitos dos fármacos , Canais de Sódio/fisiologia , Tetrodotoxina/metabolismo , Tetrodotoxina/farmacologia , XenopusRESUMO
The alpha subunit of a voltage-sensitive sodium channel characteristic of denervated rat skeletal muscle was cloned and characterized. The cDNA encodes a 2018 amino acid protein (SkM2) that is homologous to other recently cloned sodium channels, including a tetrodotoxin (TTX)-sensitive sodium channel from rat skeletal muscle (SkM1). The SkM2 protein is no more homologous to SkM1 than to the rat brain sodium channels and differs notably from SkM1 in having a longer cytoplasmic loop joining domains 1 and 2. Steady-state mRNA levels for SkM1 and SkM2 are regulated differently during development and following denervation: the SkM2 mRNA level is highest in early development, when TTX-insensitive channels predominate, but declines rapidly with age as SkM1 mRNA increases; SkM2 mRNA is not detectable in normally innervated adult skeletal muscle but increases greater than 100-fold after denervation; rat cardiac muscle has abundant SkM2 mRNA but no detectable SkM1 message. These findings suggest that SkM2 is a TTX-insensitive sodium channel expressed in both skeletal and cardiac muscle.
Assuntos
Proteínas de Membrana/genética , Músculos/metabolismo , Canais de Sódio/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Linhagem Celular , Clonagem Molecular , DNA/genética , DNA/isolamento & purificação , Proteínas de Membrana/biossíntese , Dados de Sequência Molecular , Denervação Muscular , RNA Mensageiro/análise , Ratos , Homologia de Sequência do Ácido Nucleico , Tetrodotoxina/farmacologiaRESUMO
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 microM 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 [3H]saxitoxin receptors, corresponding to TTX-resistant Na+ channels, were detected. We isolated several overlapping cDNA clones encompassing 7542 nucleotides and encoding the entire alpha subunit of a cardiac-specific 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.
Assuntos
Clonagem Molecular , Coração/crescimento & desenvolvimento , Coração/fisiologia , Proteínas de Membrana/genética , Canais de Sódio/fisiologia , Tetrodotoxina/farmacologia , Envelhecimento , Sequência de Aminoácidos , Proteínas de Anfíbios , Animais , Sequência de Bases , Northern Blotting , Encéfalo/fisiologia , Proteínas de Transporte/metabolismo , Resistência a Medicamentos , Órgão Elétrico/fisiologia , Electrophorus , Cinética , Dados de Sequência Molecular , Denervação Muscular , Músculos/fisiologia , Ratos , Homologia de Sequência do Ácido Nucleico , Canais de Sódio/efeitos dos fármacosRESUMO
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" [3H]nitrendipine receptors with Kd values corresponding well with observed physiologically effective nitrendipine concentrations. We detected two distinct populations of nitrendipine receptors in rat heart and bovine aortic membrane. A high-affinity Kd value of 0.2-0.3 nM was found, which seems to correspond to the physiologically functional state of the Ca channel in smooth muscle, since the Kd value is similar to the concentration at which nitrendipine inhibits contraction. However, in contrast to numerous other studies, we observed that the predominant component of [3H]nitrendipine binding (95-99%) had a low-affinity Kd value (235 nM). This putative low-affinity [3H]nitrendipine receptor may correspond to the physiologically functional state of the Ca channel in cardiac muscle.
Assuntos
Cálcio , Canais Iônicos/metabolismo , Músculo Liso Vascular/metabolismo , Miocárdio/metabolismo , Nitrendipino/metabolismo , Animais , Bovinos , Técnicas In Vitro , Canais Iônicos/classificação , Ensaio Radioligante , Ratos , Sarcolema/metabolismoAssuntos
Encéfalo/metabolismo , Proteínas de Transporte/metabolismo , Canais Iônicos/metabolismo , Proteínas de Membrana/metabolismo , Músculos/metabolismo , Miocárdio/metabolismo , Saxitoxina/metabolismo , Canais de Sódio , Sódio/metabolismo , Proteínas de Anfíbios , Animais , Galinhas , Cinética , RatosRESUMO
The action potential (AP) in most nerve and muscle preparations depends upon nanomolar concentrations of the neurotoxins saxitoxin (STX) and tetrodotoxin (TTX). In some excitable tissues lacking mature innervation, a toxin-resistant AP has been described by electrophysiological results. However, multiple attempts to detect corresponding toxin-resistant Na channels with radiolabelled STX and TTX have been unsuccessful. We report here the detection of Na channels with low-affinity binding of STX and TTX, accounting for 50-60% of the Na channels in rat hindlimb muscle 4-5 days after denervation.
Assuntos
Canais Iônicos/efeitos dos fármacos , Músculos/metabolismo , Saxitoxina/farmacologia , Sódio/metabolismo , Tetrodotoxina/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Resistência a Medicamentos , Técnicas In Vitro , Canais Iônicos/metabolismo , Denervação Muscular , Condução Nervosa , RatosRESUMO
Quantitative studies of 125I-labeled reovirus binding at equilibrium to several cell types was studied, including (1) murine L cell fibroblasts; (2) murine splenic T lymphocytes; (3) YAC cells, a murine lymphoma cell line; and (4) R1.1 cells, a murine thymoma cell line. Competition and saturation studies demonstrated (1) specific, saturable, high-affinity binding of reovirus types 1 and 3 to nonidentical receptors on L cell fibroblasts; (2) high-affinity binding of type 3 reovirus to murine splenic lymphocytes and R1.1 cells; (3) low-affinity binding of reovirus type 1 to lymphocytes and R1.1 cells; and (4) no significant binding of either serotype to YAC cells. Differences in the binding characteristics of the two reovirus serotypes to L cell fibroblasts were found to be a property of the viral hemagglutinin, as demonstrated using a recombinant viral clone. The equilibrium dissociation constant (Kd) for viral binding was of extremely high affinity (Kd in the range of 0.5 nM), and was slowly reversible. Experiments demonstrated temperature and pH dependence of reovirus binding and receptor modification studies using pronase, neuraminidase, and various sugars confirmed previous studies that reovirus receptors are predominantly protein in structure. The reovirus receptor site density was in the range of 2-8 X 10(4) sites/cell. These studies demonstrate that the pseudo-first-order kinetic model for ligand-receptor interactions provides a useful model for studying interactions of viral particles with membrane viral receptors. They also suggest that one cell may have distinct receptor sites for two serotypes of the same virus, and that one viral serotype may bind with different kinetics depending on the cell type.
Assuntos
Receptores Virais/fisiologia , Reoviridae/fisiologia , Animais , Linhagem Celular , Membrana Celular/microbiologia , Radioisótopos do Iodo , Cinética , Células L/microbiologia , Linfócitos/microbiologia , Linfoma , Camundongos , Camundongos Endogâmicos , Fatores de TempoRESUMO
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. The existence of two Na+ channel subtypes is proposed, with high affinity and low affinity for saxitoxin and tetrodotoxin; the significance of this classification is discussed.
Assuntos
Química Encefálica , Canais Iônicos/análise , Miocárdio/análise , Animais , Encéfalo/metabolismo , Galinhas , Canais Iônicos/metabolismo , Cinética , Proteínas de Membrana/metabolismo , Miocárdio/metabolismo , Neurônios/metabolismo , Saxitoxina , Tetrodotoxina/metabolismoRESUMO
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.
Assuntos
Axônios/metabolismo , Saxitoxina/metabolismo , Animais , Sítios de Ligação , Membrana Celular/metabolismo , Decapodiformes , Condução Nervosa , Inibição Neural , Sódio/metabolismo , TrítioRESUMO
1. Voltage-clamp studies were carried out on single rabbit myelinated nerve fibres at 14 degrees C with the method of Dodge & Frankenhaeuser (1958). 2. A method was developed to allow the ionic currents through the modal membrane to be calibrated exactly under voltage-clamp conditions by measuring the resistance of the internode through which the current was injected. 3. The ionic currents in a rabbit node of Ranvier can be resolved into two components, a sodium current and a leak current. Potassium current is almost entirely absent. 4. The sodium currents in rabbit nodes were fitted by the Hodgkin-Huxley model using m2h kinetics. The kinetics of sodium currents in a rabbit node differ from that in a frog node under similar experimental conditions in two respects: (a) inactivation is faster, tau h for rabbit being 2-3 times smaller around -50 mV; (b) the P(Na) (E) curve for mammal is shifted 10-15 mV in the hyperpolarizing direction. 5. From the kinetics of sodium current, the non-propagating rabbit action potential was reconstructed at 14 degrees C. The transient inward sodium current is responsible for the fast initial depolarization phase of the action potential, while the repolarizing phase is accounted for by leak alone. The computed shape of the action potential was in good agreement with the experimentally obtained action potential. 6. At 14 degrees C, frog and rabbit nodes with similar diameters have similar measured gNa values.
Assuntos
Fibras Nervosas Mielinizadas/fisiologia , Potenciais de Ação , Animais , Anuros , Condutividade Elétrica , Feminino , Técnicas In Vitro , Cinética , Masculino , Potenciais da Membrana , Potássio/fisiologia , Coelhos , Rana pipiens , Nós Neurofibrosos/fisiologia , Sódio/fisiologiaRESUMO
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-1, 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-1. The 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-1 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-1; the value of K was 3-8 nM.
Assuntos
Denervação Muscular , Músculos/metabolismo , Saxitoxina/metabolismo , Potenciais de Ação , Animais , Anuros , Sítios de Ligação , Condutividade Elétrica , Técnicas In Vitro , Rana pipiens , Ratos , Sódio/fisiologiaRESUMO
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 significance of these findings for saltatory conduction and in demyelinating disease is discussed.