RESUMO
Preceding the opening of the sodium channels of axon membrane there is a small outward current, gating current, that is probably associated with the molecular rearrangements that open the channels. Gating current is reversibly blocked by three procedures that block the sodium current: (i) internal perfusion with zinc ions, (ii) inactivation of sodium conductance by brief depolarization, and (iii) prolonged depolarization.
Assuntos
Axônios/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Sódio/metabolismo , Animais , Axônios/citologia , Transporte Biológico Ativo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Estimulação Elétrica , Técnicas In Vitro , Tetrodotoxina/farmacologia , Zinco/farmacologiaRESUMO
Gating currents provide a direct record of the spatial rearrangement of charges occurring within the protein of voltage-sensitive ion channels. If the elementary charges move as very brief discrete pulses of current, they will produce fluctuations in the macroscopic gating current. The variance of such fluctuations in gating currents was measured in Shaker potassium channels expressed in Xenopus oocytes with a sufficiently high recording bandwidth to estimate the magnitude and time distribution of the elementary transition charge movements. Channel activation occurred in two sequential stages. The first stage consisted of numerous, fast transitions, each moving small amounts of charge that contributed little to the fluctuation in gating current, whereas the second stage, which contributed the bulk of the fluctuation, was represented by a number of discrete, correlated transitions, one or more of which carried a charge of at least 2.4 elementary charges across the membrane field.
Assuntos
Ativação do Canal Iônico/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/metabolismo , Proteínas de Xenopus , Animais , Canal de Potássio Kv1.1 , Potenciais da Membrana , Oócitos , Mutação Puntual , Canais de Potássio/química , Canais de Potássio/genética , XenopusRESUMO
Voltage-dependent ion channels respond to changes in the membrane potential by means of charged voltage sensors intrinsic to the channel protein. Changes in transmembrane potential cause movement of these charged residues, which results in conformational changes in the channel. Movements of the charged sensors can be detected as currents known as gating currents. Measurement of the gating currents of the Drosophila Shaker potassium channel indicates that the charge on the voltage sensor of the channels is progressively immobilized by prolonged depolarizations. The charge is not immobilized in a mutant of the channel that lacks inactivation. These results show that the region of the molecule responsible for inactivation interacts, directly or indirectly, with the voltage sensor to prevent the return of the charge to its original position. The gating transitions between closed states of the channel appear not to be independent, suggesting that the channel subunits interact during activation.
Assuntos
Ativação do Canal Iônico , Canais de Potássio/fisiologia , Sequência de Aminoácidos , Animais , Drosophila/fisiologia , Ativação do Canal Iônico/efeitos dos fármacos , Cinética , Mutagênese Sítio-Dirigida , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/genética , Tetraetilamônio , Compostos de Tetraetilamônio/farmacologia , XenopusRESUMO
The delayed rectifier K+ channel of the squid axon undergoes a series of modifications in its kinetic and conductive parameters when it is phosphorylated as the result of shifts in its voltage-dependent parameters. These effects can be interpreted as due to electrostatic interaction between the voltage sensor of the channel and the transferred phosphate from ATP. Using different concentrations of intracellular Mg2+, we determined the density of surface charges seen by the K+ channel voltage sensor before and after phosphorylation. Values for the surface charge density in the cytoplasmic side of the membrane were between 1/350 and 1/250 e-/A2 in the absence of ATP and between 1/160 and 1/155 e-/A2 under phosphorylating conditions. Incorporation of a surface potential into a kinetic model for the delayed rectifier channel can predict quantitatively phosphorylation-like changes in K+ currents. These results provide evidence for the importance of electrostatic interactions as one of the mechanisms by which phosphorylation modulates the behavior of voltage-dependent channels.
Assuntos
Axônios/metabolismo , Ativação do Canal Iônico , Canais de Potássio/fisiologia , Animais , Decapodiformes , Eletrofisiologia , Modelos Neurológicos , Fosforilação , Canais de Potássio/metabolismoRESUMO
We examined voltage-dependent conformational changes in three specific regions of the Shaker potassium channel with site-directed fluorescent labeling: the fourth transmembrane segment (S4), the second transmembrane segment (S2), and the putative pore region. The fluorescence changes displayed distinctive properties that correlate with gating, activation, and slow inactivation of the channel. The fluorescence signals measured near the S2 and S4 segments suggest that the S2 segment may undergo voltage-sensitive conformational changes that precede those in the S4 segment. In contrast, fluorescence changes in the pore correlated with the voltage dependence and time course of ionic activation and slow inactivation. Spectroscopy indicated that the mechanism of fluorescence change involves voltage-dependent quenching of the probe in an aqueous environment by other parts of the protein.
Assuntos
Canais de Potássio/química , Canais de Potássio/fisiologia , Animais , Drosophila , Proteínas de Drosophila , Condutividade Elétrica , Eletrofisiologia , Fluorescência , Corantes Fluorescentes , Ativação do Canal Iônico/fisiologia , Cinética , Modelos Biológicos , Conformação Molecular , Oócitos/metabolismo , Superfamília Shaker de Canais de Potássio , XenopusRESUMO
We have cloned a Kv2 potassium channel from squid optic lobe termed sqKv2. Multiple overlapping sqKv2 cDNA clones differed from one another at specific positions by purine transitions. To test whether the purine transitions were generated by RNA editing, we compared a 360 nucleotide genomic sequence with corresponding cDNA sequences (encoding S4-S6) isolated from individual animals and lying on a single gene and exon. cDNA sequences differed from genomic sequence at 17 positions, resulting in 28 unique sequences. There was invariantly an adenosine in the genomic sequence and a guanosine in the edited cDNA sequences. Two of the edits altered the rates of channel closure and slow inactivation. These results extend selective RNA editing to invertebrate taxa and represents a novel mechanism for the posttranscriptional modulation of voltage-gated ion channels.
Assuntos
Decapodiformes/genética , Canais de Potássio/genética , Edição de RNA/fisiologia , RNA Mensageiro/metabolismo , Adenina , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar , Eletrofisiologia , Éxons/fisiologia , Expressão Gênica/fisiologia , Genótipo , Guanina , Dados de Sequência Molecular , Mutagênese/fisiologia , Lobo Óptico de Animais não Mamíferos/química , Canais de Potássio/metabolismo , Transcrição Gênica/fisiologiaRESUMO
In voltage-dependent ion channels, pore opening is initiated by electrically driven movements of charged residues, and this movement generates a gating current. To examine structural rearrangements in the Shaker K+ channel, basic residues R365 and R368 in the S4 segment were replaced with histidine, and gating currents were recorded. Changes in gating charge displacement with solvent pH reveal voltage-dependent changes in exposure of the histidine to solvent protons. This technique directly monitors accessibility changes during gating, probes the environment even in confined locations, and introduces minimal interference of gating charge motion. The results indicate that charges 365 and 368 traverse the entire electric field during gating. The remarkable implication of the successive exposure of histidine to each side of the membrane is that in a pH gradient, the voltage sensor transports protons.
Assuntos
Canais de Potássio/fisiologia , Substituição de Aminoácidos , Animais , Sequência de Bases , Sequência Consenso , Histidina , Concentração de Íons de Hidrogênio , Ativação do Canal Iônico , Cinética , Meglumina/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Modelos Biológicos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Mutação Puntual , Canais de Potássio/química , Superfamília Shaker de Canais de PotássioRESUMO
In voltage-dependent ion channels, a voltage sensor region is responsible for channel activation and an aqueous pore is responsible for ion conduction. These two processes have been traditionally considered to be independent. We describe here a mutation in the putative pore region (W434F) that completely abolishes ion conduction without affecting the gating charge of the channel. Gating currents in the nonconductive mutant were found to be identical in their kinetic and steady-state properties to those in conductive channels. Gating current measurements could be performed without subtracting pulses and in the presence of normal physiological solutions. Application of internal tetraethylammonium (an open channel blocker) induced Off charge immobilization for large depolarizations, suggesting that the internal tetraethylammonium-binding site becomes available upon depolarization. We concluded that for this mutant, although the conduction pathway is not functional, the channel can still undergo the closed-open conformation in response to voltage changes.
Assuntos
Drosophila/genética , Drosophila/metabolismo , Ativação do Canal Iônico , Mutação , Canais de Potássio/fisiologia , Animais , Condutividade Elétrica , Canais de Potássio/efeitos dos fármacos , Tetraetilamônio , Compostos de Tetraetilamônio/farmacologiaRESUMO
The activation of Shaker K+ channels is steeply voltage dependent. To determine whether conserved charged amino acids in putative transmembrane segments S2, S3, and S4 contribute to the gating charge of the channel, the total gating charge movement per channel was measured in channels containing neutralization mutations. Of eight residues tested, four contributed significantly to the gating charge: E293, an acidic residue in S2, and R365, R368, and R371, three basic residues in the S4 segment. The results indicate that these residues are a major component of the voltage sensor. Furthermore, the S4 segment is not solely responsible for gating charge movement in Shaker K+ channels.
Assuntos
Potenciais da Membrana/fisiologia , Canais de Potássio/fisiologia , Animais , XenopusRESUMO
Using site-directed fluorescent labeling, we examined conformational changes in the S4 segment of each domain of the human skeletal muscle sodium channel (hSkM1). The fluorescence signals from S4 segments in domains I and II follow activation and are unaffected as fast inactivation settles. In contrast, the fluorescence signals from S4 segments in domains III and IV show kinetic components during activation and deactivation that correlate with fast inactivation and charge immobilization. These results indicate that in hSkM1, the S4 segments in domains III and IV are responsible for voltage-sensitive conformational changes linked to fast inactivation and are immobilized by fast inactivation, while the S4 segments in domains I and II are unaffected by fast inactivation.
Assuntos
Canais de Sódio/genética , Canais de Sódio/fisiologia , Animais , Eletroquímica , Feminino , Humanos , Ativação do Canal Iônico/fisiologia , Cinética , Microscopia de Fluorescência , Modelos Biológicos , Conformação Molecular , Mutação/fisiologia , Oócitos , Canais de Sódio/química , Fatores de Tempo , XenopusRESUMO
We report the measurement of a Cai-activated membrane current in dialyzed squid axon under membrane potential control with a low-noise voltage clamp. Two additional voltage clamp systems were used to clamp the external guard plates to a value that prevented the establishment of potential differences between the central and lateral compartments of the experimental chamber. This reduced to a minimum the contribution of membrane currents generated at the axon ends to the current measured in the central pool. This latter current was reduced by using internal and external solutions designed to diminish at a maximum membrane currents, while maintaining the conditions for optimal operation of the Na+-Ca2+ exchange. Thus TTX was used to block Na+ channels and prolonged exposure to K+-free media was used to eliminate K+ conductance. The maximum concentration of external sodium was 200 mM. The addition of fixed amounts of free ionic calcium to the internal solution, activated a current whose direction and magnitude depended on the thermodynamic driving forces for calcium and sodium. When the experimental conditions determined an inwardly directed current, this depended on the presence of external sodium, and lithium could not substitute for it. The Cai-activated current, was blocked by external lanthanum and showed a high temperature dependence. In experiments in which the reversal potential was measured for the Cai-activated current, it was found to be strikingly similar to the value calculated according to Er = 3ENa - 2ECa, suggesting that the current is the electrical manifestation of the Na+-Ca2+ exchange operating with an stoichiometry of 3Na+:1Ca2+.
Assuntos
Axônios/fisiologia , Cálcio/metabolismo , Sódio/metabolismo , Animais , Axônios/efeitos dos fármacos , Cálcio/farmacologia , Membrana Celular/fisiologia , Decapodiformes , Ácido Egtázico/farmacologia , Condutividade Elétrica , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/fisiologia , Lantânio/farmacologia , Potenciais da MembranaRESUMO
Gating currents were recorded at 11 degrees C in cell-attached and inside-out patches from the innervated membrane of Electrophorus main organ electrocytes. With pipette tip diameters of 3-8 microns, maximal charge measured in patches ranged from 0.74 to 7.19 fC. The general features of the gating currents are similar to those from the squid giant axon. The steady-state voltage dependence of the ON gating charge was characterized by an effective valence of 1.3 +/- 0.4 and a midpoint voltage of -56 +/- 7 mV. The charge vs. voltage relation lies approximately 30 mV negative to the channel open probability curve. The ratio of the time constants of the OFF gating current and the Na current was 2.3 at -120 mV and equal at -80 mV. Charge immobilization and Na current inactivation develop with comparable time courses and have very similar voltage dependences. Between 60 and 80% of the charge is temporarily immobilized by inactivation.
Assuntos
Órgão Elétrico/fisiologia , Electrophorus/fisiologia , Ativação do Canal Iônico/fisiologia , Canais de Sódio/fisiologia , Potenciais de Ação/fisiologia , Animais , Eletrodos , Fatores de TempoRESUMO
One measure of the voltage dependence of ion channel conductance is the amount of gating charge that moves during activation and vice versa. The limiting slope method, introduced by Almers (Almers, W. 1978. Rev. Physiol. Biochem. Pharmacol. 82:96-190), exploits the relationship of charge movement and voltage sensitivity, yielding a lower limit to the range of single channel gating charge displacement. In practice, the technique is plagued by low experimental resolution due to the requirement that the logarithmic voltage sensitivity of activation be measured at very low probabilities of opening. In addition, the linear sequential models to which the original theory was restricted needed to be expanded to accommodate the complexity of mechanisms available for the activation of channels. In this communication, we refine the theory by developing a relationship between the mean activation charge displacement (a measure of the voltage sensitivity of activation) and the gating charge displacement (the integral of gating current). We demonstrate that recording the equilibrium gating charge displacement as an adjunct to the limiting slope technique greatly improves accuracy under conditions where the plots of mean activation charge displacement and gross gating charge displacement versus voltage can be superimposed. We explore this relationship for a wide variety of channel models, which include those having a continuous density of states, nonsequential activation pathways, and subconductance states. We introduce new criteria for the appropriate use of the limiting slope procedure and provide a practical example of the theory applied to low resolution simulation data.
Assuntos
Ativação do Canal Iônico , Canais Iônicos/fisiologia , Modelos Biológicos , Eletrofisiologia , TermodinâmicaRESUMO
When attached to specific sites near the S4 segment of the nonconducting (W434F) Shaker potassium channel, the fluorescent probe tetramethylrhodamine maleimide undergoes voltage-dependent changes in intensity that correlate with the movement of the voltage sensor (Mannuzzu, L.M., M.M. Moronne, and E.Y. Isacoff. 1996. Science. 271:213-216; Cha, A., and F. Bezanilla. 1997. Neuron. 19:1127-1140). The characteristics of this voltage-dependent fluorescence quenching are different in a conducting version of the channel with a different pore substitution (T449Y). Blocking the pore of the T449Y construct with either tetraethylammonium or agitoxin removes a fluorescence component that correlates with the voltage dependence but not the kinetics of ionic activation. This pore-mediated modulation of the fluorescence quenching near the S4 segment suggests that the fluorophore is affected by the state of the external pore. In addition, this modulation may reflect conformational changes associated with channel opening that are prevented by tetraethylammonium or agitoxin. Studies of pH titration, collisional quenchers, and anisotropy indicate that fluorophores attached to residues near the S4 segment are constrained by a nearby region of protein. The mechanism of fluorescence quenching near the S4 segment does not involve either reorientation of the fluorophore or a voltage-dependent excitation shift and is different from the quenching mechanism observed at a site near the S2 segment. Taken together, these results suggest that the extracellular portion of the S4 segment resides in an aqueous protein vestibule and is influenced by the state of the external pore.
Assuntos
Ativação do Canal Iônico/fisiologia , Canais de Potássio/química , Canais de Potássio/genética , Animais , Anisotropia , Corantes Fluorescentes , Concentração de Íons de Hidrogênio , Ativação do Canal Iônico/efeitos dos fármacos , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Mutagênese Sítio-Dirigida/fisiologia , Oócitos/fisiologia , Técnicas de Patch-Clamp , Conformação Proteica , Venenos de Escorpião/farmacologia , Superfamília Shaker de Canais de Potássio , Tetraetilamônio/farmacologiaRESUMO
Patch pipettes were used to record the current arising from small populations of sodium channels in voltage-clamped cut-open squid axons. The current fluctuations associated with the time-variant sodium conductance were analyzed with nonstationary statistical techniques in order to obtain an estimate for the conductance of a single sodium channel. The results presented support the notion that the open sodium channel in the squid axon has only one value of conductance, 3.5 pS.
Assuntos
Axônios/metabolismo , Decapodiformes/metabolismo , Canais Iônicos/metabolismo , Sódio/metabolismo , Animais , Eletrofisiologia , RatosRESUMO
The presence of internal Mg-ATP produced a number of changes in the K conductance of perfused giant axons of squid. For holding potentials between -40 and -50 mV, steady-state K conductance increased for depolarizations to potentials more positive than approximately -15 mV and decreased for smaller depolarizations. The voltage dependencies of both steady-state activation and inactivation also appears shifted toward more positive potentials. Gating kinetics were affected by internal ATP, with the activation time constant slowed and the characteristic delay in K conductance markedly enhanced. The rate of deactivation also was hastened during perfusion with ATP. Internal ATP affected potassium channel gating currents in similar ways. The voltage dependence of gating charge movement was shifted toward more positive potentials and the time constants of ON and OFF gating current also were slowed and hastened, respectively, in the presence of ATP. These effects of ATP on the K conductance occurred when no exogenous protein kinases were added to the internal solution and persisted even after removing ATP from the internal perfusate. Perfusion with a solution containing exogenous alkaline phosphatase reversed the effects of ATP. These results provide further evidence that the effects of ATP on the K conductance are a consequence of a phosphorylation reaction mediated by a kinase present and active in perfused axons. Phosphorylation appears to alter the K conductance of squid giant axons via a minimum of two mechanisms. First, the voltage dependence of gating parameters are shifted toward positive potentials. Second, there is an increase in the number of functional closed states and/or a decrease in the rates of transition between these states of the K channels.
Assuntos
Trifosfato de Adenosina/farmacologia , Axônios/fisiologia , Condução Nervosa/fisiologia , Canais de Potássio/fisiologia , Animais , Axônios/efeitos dos fármacos , Decapodiformes , Eletrofisiologia , Técnicas In Vitro , Condução Nervosa/efeitos dos fármacos , Fosforilação , Canais de Potássio/efeitos dos fármacosRESUMO
The sodium current (I(Na)) that develops after step depolarization of a voltage clamped squid axon is preceded by a transient outward current that is closely associated with the opening of the activation gates of the Na pores. This "gating current" is best seen when permeant ions (Na and K) are replaced by relatively impermeant ones, and when the linear portion of capacitative current is eliminated by adding current from positive steps to that from exactly equal negative ones. During opening of the Na pores gating current is outward, and as the pores close there is an inward tail of current that decays with approximately the same time-course as I(Na) recorded in Na-containing medium. Both outward and inward gating current are unaffected by tetrodotoxin (TTX). Gating current is capacitative in origin, the result of relatively slow reorientation of charged or dipolar molecules in a suddenly altered membrane field. Close association with the Na activation process is clear from the time-course of gating current, and from the fact that three procedures that reversibly block I(Na) also block gating current: internal perfusion with Zn(2+), prolonged depolarization of the membrane, and inactivation of I(Na) with a short positive prepulse.
Assuntos
Axônios/metabolismo , Permeabilidade da Membrana Celular , Sódio/metabolismo , Animais , Decapodiformes , Eletrofisiologia , Técnicas In Vitro , Métodos , Tetrodotoxina/farmacologia , ZincoRESUMO
In squid axons, internally applied ATP potentiates the magnitude of the potassium conductance and slows down its activation kinetics. This effect was characterized using internally dialyzed axons under voltage-clamp conditions. Both amplitude potentiation and kinetic slow-down effects are very selective towards ATP, other nucleotides like GTP and ITP are ineffective in millimolar concentrations. The current potentiation Km for ATP is near 10 microM with no further effects for concentrations greater than 100 microM. ATP effect is most likely produced via a phosphorylative reaction because Mg ion is an obligatory requirement and nonhydrolyzable ATP analogues are without effect. In the presence of ATP, the K current presents more delay, resembling a Cole-Moore effect due to local hyperpolarization of the channel. ATP effect induces a 10-20 mV shift in both activation and inactivation parameters towards more depolarized potentials. As a consequence of this shift, conductance-voltage curves with and without ATP cross at approximately -40 mV. This result is consistent with the hyperpolarization observed with ATP depletion, which is reversed by ATP addition. At potentials around the resting value, addition of ATP removes almost completely K current slow inactivation. It is suggested that a change in the amount of the slow inactivation is responsible for the differences in current amplitude with and without ATP, possibly as a consequence of the additional negative charge carried by the phosphate group. However, a modification of the local potential is not enough to explain completely the differences under the two conditions.
Assuntos
Trifosfato de Adenosina/farmacologia , Axônios/metabolismo , Canais de Potássio/metabolismo , Animais , Axônios/efeitos dos fármacos , Decapodiformes , Técnicas In Vitro , Fosforilação , Canais de Potássio/efeitos dos fármacosRESUMO
The voltage sensor of the Shaker potassium channel is comprised mostly of positively charged residues in the putative fourth transmembrane segment, S4 (Aggarwal, S.K., and R. MacKinnon. 1996. Neuron. 16:1169-1177; Seoh, S.-A., D. Sigg, D.M. Papazian, and F. Bezanilla. 1996. Neuron. 16:1159-1167). Movement of the voltage sensor in response to a change in the membrane potential was examined indirectly by measuring how the accessibilities of residues in and around the sensor change with voltage. Each basic residue in the S4 segment was individually replaced with a histidine. If the histidine tag is part of the voltage sensor, then the gating charge displaced by the voltage sensor will include the histidine charge. Accessibility of the histidine to the bulk solution was therefore monitored as pH-dependent changes in the gating currents evoked by membrane potential pulses. Histidine scanning mutagenesis has several advantages over other similar techniques. Since histidine accessibility is detected by labeling with solution protons, very confined local environments can be resolved and labeling introduces minimal interference of voltage sensor motion. After histidine replacement of either residue K374 or R377, there was no titration of the gating currents with internal or external pH, indicating that these residues do not move in the transmembrane electric field or that they are always inaccessible. Histidine replacement of residues R365, R368, and R371, on the other hand, showed that each of these residues traverses entirely from internal exposure at hyperpolarized potentials to external exposure at depolarized potentials. This translocation enables the histidine to transport protons across the membrane in the presence of a pH gradient. In the case of 371H, depolarization drives the histidine to a position that forms a proton pore. Kinetic models of titrateable voltage sensors that account for proton transport and conduction are presented. Finally, the results presented here are incorporated into existing information to propose a model of voltage sensor movement and structure.
Assuntos
Histidina/genética , Ativação do Canal Iônico/fisiologia , Canais de Potássio , Animais , Concentração de Íons de Hidrogênio , Potenciais da Membrana/fisiologia , Modelos Biológicos , Mutagênese/fisiologia , Oócitos/fisiologia , Técnicas de Patch-Clamp , Potássio/metabolismo , Canais de Potássio/química , Canais de Potássio/genética , Canais de Potássio/metabolismo , Estrutura Quaternária de Proteína , Prótons , Superfamília Shaker de Canais de Potássio , XenopusRESUMO
Inactivation of sodium conductance has been studied in squid axons with voltage clamp techniques and with the enzyme pronase which selectively destroys inactivation. Comparison of the sodium current before and after pronase treatment shows a lag of several hundred microseconds in the onset of inactivation after depolarization. This lag can of several hundred microseconds in the onset of inactivation after polarization. This lag can also be demonstrated with double-pulse experiments. When the membrane potential is hyperpolarized to -140 mV before depolarization, both activation and inactivation are delayed. These findings suggest that inactivation occurs only after activation are delayed. These findings suggest that inactivation occurs only after activation; i.e. that the channels must open before they can inactivate. The time constant of inactivation measured with two pulses (tau(c)) is the same as the one measured from the decay of the sodium current during a single pulse (tau(h)). For large depolarizations, steady-state inactivation becomes more incomplete as voltage increases; but it is relatively complete and appears independent of voltage when determined with a two- pulse method. This result confirms the existence of a second open state for Na channels, as proposed by Chandler and Meves (1970. J. Physiol. [Lond.]. 211:653-678). The time constant of recovery from inactivation is voltage dependent and decreases as the membrane potential is made more negative. A model for Na channels is presented which has voltage-dependent transitions between the closed and open states, and a voltage-independent transition between the open and the inactivated state. In this model the voltage dependence of inactivation is a consequence of coupling to the activation process.