GABA(B) presynaptically modulates suprachiasmatic input to hypothalamic paraventricular magnocellular neurons

This study used whole cell patch clamp recordings in rat hypothalamic slice preparations to evaluate the effects of GABA(B) receptor activation on GABA(A)-mediated inhibitory postsynaptic currents (IPSCs) in paraventricular nucleus magnocellular neurons evoked by electrical stimulation in the suprachiasmatic nucleus (SCN). Baclofen induced a dose-dependent (1-10 microM) and reversible reduction in SCN-evoked IPSC amplitude (11/11 cells), blockable with 2-hydroxysaclofen (300 microM; 3/3 cells). IPSCs displayed paired-pulse depression (PPD), attenuated by both baclofen and 2-hydroxysaclofen, but neither altered resting membrane conductances or IPSC time constants of decay. Baclofen induced a significant dose-dependent (1-100 microM) reduction in frequency, but not amplitude, of spontaneous IPSCs and miniature IPSCs, reversible with 2-hydroxysaclofen pretreatment. Baclofen effects and PPD persisted in slices pretreated with pertussis toxin (PTX) and N-ethylmaleimide, implying that these GABA(B) receptors are coupled to PTX-insensitive G proteins. Responses were unaltered by barium (2 mM) or nimodipine, ruling out involvement of K(+) channels and L-type Ca(2+) channels. Thus pre- and postsynaptic GABA(B) and GABA(A) receptors participate in SCN entrainment of paraventricular neurosecretory neurons.     

Asymmetric electrostatic effects on the gating of rat brain sodium channels in planar lipid membranes.

The effects of ionic strength (10-1,000 mM) on the gating of batrachotoxin-activated rat brain sodium channels were studied in neutral and in negatively charged lipid bilayers. In neutral bilayers, increasing the ionic strength of the extracellular solution, shifted the voltage dependence of the open probability (gating curve) of the sodium channel to more positive membrane potentials. On the other hand, increasing the intracellular ionic strength shifted the gating curve to more negative membrane potentials. Ionic strength shifted the voltage dependence of both opening and closing rate constants of the channel in analogous ways to its effects on gating curves. The voltage sensitivities of the rate constants were not affected by ionic strength. The effects of ionic strength on the gating of sodium channels reconstituted in negatively charged bilayers were qualitatively the same as in neutral bilayers. However, important quantitative differences were noticed: in low ionic strength conditions (10-150 mM), the presence of negative charges on the membrane surface induced an extra voltage shift on the gating curve of sodium channels in relation to neutral bilayers. It is concluded that: (a) asymmetric negative surface charge densities in the extracellular (1e-/533A2) and intracellular (1e-/1,231A2) sides of the sodium channel could explain the voltage shifts caused by ionic strength on the gating curve of the channel in neutral bilayers. These surface charges create negative electric fields in both the extracellular and intracellular sides of the channel. Said electric fields interfere with gating charge movements that occur during the opening and closing of sodium channels; (b) the voltage shifts caused by ionic strength on the gating curve of sodium channels can be accounted by voltage shifts in both the opening and closing rate constants; (c) net negative surface charges on the channel's molecule do not affect the intrinsic gating properties of sodium channels but are essential in determining the relative position of the channel's gating curve; (d) provided the ionic strength is below 150 mM, the gating machinery of the sodium channel molecule is able to sense the electric field created by surface changes on the lipid membrane. I propose that during the opening and closing of sodium channels, the gating charges involved in this process are asymmetrically displaced in relation to the plane of the bilayer. Simple electrostatic calculations suggest that gating charge movements are influenced by membrane electrostatic potentials at distances of 48 and 28 A away from the plane of the membrane in the extracellular sides of the channel, respectively.     

Modification of sodium channel gating by lanthanum. Some effects that cannot be explained by surface charge theory

In clonal pituitary (GH3) cells we studied the changes in sodium channel gating caused by substitution of La3+ for Ca2+ ion. Gating of sodium channels was simplified by using intracellular papain to remove inactivation. To quantify La effects, we empirically fitted closing and the late phase of opening of the channels with single exponentials, determined the opening (a) and closing (b) rate, and plotted these rates as a function of Vm (membrane voltage). The midpoint of the fraction open-Vm curve was also determined. Changing from Ca to La shifted the curves for these three measures of Na channel gating along the voltage axis and changed their shape somewhat. Surface charge theory, in the form usually presented, predicts equal shifts of all three curves, with no change in shape. We found, however, that the shift for each of the measurements was different. 2 mM La, for example, shifted opening kinetics by +52 mV (i.e., 52 mV must be added to the depolarization to make activation in 2 mM La as fast as in 2 mM Ca), the fraction open voltage curve by +42.5 mV, and the closing rate curve by +28 mV. The shift was an almost linear function of log [La] for each of the measures. The main finding is that changing from 2 mM Ca to 10 microM La causes a positive shift of the opening rate and fraction open curves, but a negative shift of the closing rate curve. The opposite signs of the two effects cannot be explained in terms of surface charge theory. We briefly discuss some alternatives to this theory.     

GABA-Mediated Inhibition in Cultured Neurons of the Rat Brain Cortex

In cultured pyramidal neurons of the rat brain cortex, we recorded (in the whole-cell configuration) postsynaptic currents (PSC) evoked by direct electrical microstimulation of an axon of the interneuron adjacent to the pyramidal cell. Application of 5 M bicuculline rapidly, entirely, and reversibly blocked these currents. Linear changes in the holding potential on the membrane of the postsynaptic cell resulted in linear changes in the amplitude of averaged currents. The currents underwent reversion when the holding potential was –16 mV, which was close to the reversal potential for Cl^- ions at their respective concentrations in the extra- and intracellular solutions. We conclude that the recorded currents are inhibitory PSC (IPSC) mediated by GABA release. The amplitudes of the recorded currents varied from a measurable minimum (8 pA) to more than 150 pA at a holding potential on the postsynaptic cell membrane of –80 mV. Times to peak of the high- and low-amplitude currents showed no significant differences, being about 6.4 msec on average. Decays of the current could be satisfactorily approximated by a monoexponential function with a mean time constant of 17 msec. The time constants of IPSC decay were distributed accordingly to the Gaussian law. In some cases, the amplitude distributions of IPSC were unimodal ((with a rightward asymmetry), but in most cases they were clearly polymodal. The amplitude distribution can be described by the sum of several Gaussian distributions; the distance between modes of the Gaussians was 25 ± 6 pA, on average. The obtained estimates of the amplitude of monoquantal GABA-induced IPSC in neurons of the brain cortex allow us to conclude that in various CNS regions the dimension of the vesicles in GABA-ergic synapses formed by inhibitory interneurons is identical.     

Sodium channels in cultured chick dorsal root ganglion neurons

Isolated Na currents were studied in cultured chick sensory neurons using the patch clamp technique. On membrane depolarization, whole cell currents showed the typical transient and voltage-dependent time course as in nerve fibres. Na currents appeared at about-40 mV and reached maximum amplitude at around-10 mV. At low voltages (-30 to 0 mV), their turning-on was sigmoidal and inactivation developed exponentially. The ratio of inactivation time constants was found to be smaller than in squid axons and comparable to that of mammalian nodes of Ranvier. Peak conductance and steady-state inactivation were strongly voltage-dependent, with maximum slopes at-17 and-40 mV, respectively. The reversal potential was close to the Nernst equilibrium potential, indicating a high degree of ion-selectivity for the channel. Addition of 3M TTX, or replacement of Na by Choline in the external bath, abolished these currents. Internal pronase (1 mg/ml) and N-bromoacetamide (0.4 mM) made inactivation incomplete, with little effect on its rate of decay.Single Na channel currents were studied in outside-out membrane patches, at potentials between-50 and-20 mV. Their activation required large negative holding potentials (-90 mV). They were fully blocked by addition of TTX (3 M) to the external bath. At-40 mV their mean open time was about 2ms and the amplitude distribution could be fitted by a single Gaussian curve, indicating the presence of a homogeneous population of channels with a conductance of 11±2 pS. Probability of opening increased and latency to first opening decreased with increasing depolarization. Inactivation of the channel became faster with stronger depolarizations, as measured from the inactivation time course of sample averages. Internal pronase (0.1 mg/ml) produced effects on inactivation comparable to those on whole cell currents. Openings of the channel had a tendency to occur in bursts and showed little inactivation during pulses of 250 ms duration. The open lifetime of the channel at low potentials (-50,-40 mV) was only three times larger than in control patches, suggesting that Na channels in chick sensory neurons can close several times before entering an inactivating absorbing state.     

Kinetic characteristics of inhibitory postsynaptic currents in cultured chick embryo spinal cord neurons

Decay of inhibitory postsynaptic currents (IPSC) was analyzed in dissociated culture of chick embryo spinal cord. Differences in the kinetic characteristics of low-amplitude and giant IPSC were revealed. Decay of currents in the first group was single-exponential, while decay in the second group was double-exponential. The time constant of single-exponential current decay increased during membrane depolarization and decreased during rise in temperature of the solution. Decay of the double-exponential currents depended little on potential, while temperature changes acted only on its slow component. Strychnine in submaximum concentrations produced not only a decrease in amplitude of giant IPSC, but also a deceleration of decay due to the slow component. The regularity of these phenomena suggests that decay of giant IPSC, as distinguished from that of low-amplitude currents, is determined by removal of transmitter from the synaptic cleft.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the USSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 4, pp. 427–435, July–August, 1991.     

Two novel residues in M2 of the gamma-aminobutyric acid type A receptor affecting gating by GABA and picrotoxin affinity

An amino acid residue was found in M2 of gamma-aminobutyric acid (GABA) type A receptors that has profound effects on the binding of picrotoxin to the receptor and therefore may form part of its binding pocket. In addition, it strongly affects channel gating. The residue is located N-terminally to residues suggested so far to be important for channel gating. Point mutated alpha1beta(3) receptors were expressed in Xenopus oocytes and analyzed using the electrophysiological techniques. Coexpression of the alpha(1) subunit with the mutated beta(3) subunit beta(3)L253F led to spontaneous picrotoxin-sensitive currents in the absence of GABA. Nanomolar concentrations of GABA further promoted channel opening. Upon washout of picrotoxin, a huge transient inward current was observed. The reversal potential of the inward current was indicative of a chloride ion selectivity. The amplitude of the inward current was strongly dependent on the picrotoxin concentration and on the duration of its application. There was more than a 100-fold decrease in picrotoxin affinity. A kinetic model is presented that mimics the gating behavior of the mutant receptor. The point mutation in the neighboring residue beta(3)A252V resulted in receptors that displayed an about 6-fold increased apparent affinity to GABA and an about 10-fold reduced sensitivity to picrotoxin.     

A conductance decrease after application of GABA to crayfish muscle fibers

Relaxations after voltage steps of membrane current elicited by superfusion with low concentrations of GABA (up to 50 mumol/l) were measured. In many preparations, a conductance decrease due to GABA was observed. The response to GABA was shown to consist of two major components: the well known opening of synaptic chloride channels, and the closing of previously open channels, presumably permeable to K+ ions. The latter component could not be blocked by picrotoxin.     

Kinetic properties and inactivation of the gating currents of sodium channels in squid axon.

Associated with the opening and closing of the sodium channels of nerve membrane is a small component of capacitative current, the gating current. After termination of a depolarizing step the gating current and sodium current decay with similar time courses. Both currents decay more rapidly at relatively negative membrane voltages than at positive ones. The gating current that flows during a depolarizing step is diminished by a pre-pulse that inactivates the sodium permeability. A pre-pulse has no effect after inactivation has been destroyed by internal perfusion with the proteolytic enzyme pronase. Gating charge (considered as positive charge) moves outward during a positive voltage step, with voltage dependent kinetics. The time constant of the outward gating current is a maximum at about minus 10 mV, and has a smaller value at voltages either more positive or negative than this value.     

Cyclic AMP-dependent protein kinase decreases GABAA receptor current in mouse spinal neurons

GABA, the major inhibitory neurotransmitter in the mammalian brain, binds to GABAA receptors, which form chloride ion channels. The predicted structure of the GABAA receptor places a consensus phosphorylation site for cAMP-dependent protein kinase (PKA) on an intracellular domain of the channel. Phosphorylation by various protein kinases has been shown to alter the activity of certain ligand- and voltage-gated ion channels. We have examined the role of phosphorylation by the catalytic subunit of PKA in the regulation of GABAA receptor channel function using whole-cell and excised outside-out patch-clamp techniques. Inclusion of the catalytic subunit of PKA in the recording pipettes significantly reduced GABA-evoked whole-cell and single-channel chloride currents. Both heat inactivation of PKA and addition of the specific protein kinase inhibitor peptide prevented the reduction of GABA-evoked currents by PKA. Neither mean channel open time nor channel conductance was affected by PKA. The reduction in GABA receptor current by PKA was primarily due to a reduction in channel opening frequency.     

Rate of opening of a population of Na channels in frog skeletal muscle fibers

Linear Systems convolution analysis of muscle sodium currents was used to predict the opening rate of sodium channels as a function of time during voltage clamp pulses. If open sodium channel lifetimes are exponentially distributed, the channel opening rate corresponding to a sodium current obtained at any particular voltage, can be analytically obtained using a simple equation, given single channel information about the mean open-channel lifetime and current.Predictions of channel opening rate during voltage clamp pulses show that sodium channel inactivation arises coincident with a decline in channel opening rate.Sodium currents pharmacologically modified with Chloramine-T treatment so that they do not inactivate, show a predicted sustained channel opening rate.Large depolarizing voltage clamp pulses produce channel opening rate functions that resemble gating currents.The predicted channel opening rate functions are best described by kinetic models for Na channels which confer most of the charge movement to transitions between closed states.Comparisons of channel opening rate functions with gating currents suggests that there may be subtypes of Na channel with some contributing more charge movement per channel opening than others.Na channels open on average, only once during the transient period of Na activation and inactivation.After transiently opening during the activation period and then closing by entering the inactivated state, Na channels reopen if the voltage pulse is long enough and contribute to steady-state currents.The convolution model overestimates the opening rate of channels contributing to the steady-state currents that remain after the transient early Na current has subsided.     

GABA receptors on the cell-body membrane of an identified insect motor neuron

The pharmacology of a gamma-aminobutyric acid (GABA) receptor on the cell body of an identified motor neuron of the cockroach (Periplaneta americana) was investigated by current-clamp and voltage-clamp methods. Iontophoretic application of GABA increased membrane conductance to chloride ions, and prolonged application resulted in desensitization. Hill coefficients, determined from dose-response data, indicated that binding of at least two GABA molecules was required to activate the chloride channel. Differences between vertebrate GABAA receptors and insect neuronal GABA receptors were detected. For the GABA receptor of motor neuron Df, the following rank order of potency was observed: isoguvacine greater than muscimol greater than or equal to GABA greater than 3-aminopropanesulphonic acid. The GABAB receptor agonist baclofen was inactive. Of the potent vertebrate GABA receptor antagonists (bicuculline, pitrazepin, RU5135 and picrotoxin), only picrotoxin (10(-7) M) produced a potent, reversible block of the response to GABA of motor neuron Df. Both picrotoxinin and picrotin also blocked GABA-induced currents. Bicuculline hydrochloride (10(-4) M) and bicuculline methiodide (10(-4) M) were both ineffective when applied at resting membrane potential (-65 mV), although at hyperpolarized levels partial block of GABA-induced current was sometimes observed. Pitrazepin (10(-4) M) caused a partial, voltage-independent block of GABA-induced current. The steroid derivative RU5135 was inactive at 10(-5) M. In contrast to the potent competitive blockade of vertebrate GABAA receptors by bicuculline, pitrazepin and RU5135, none of the weak antagonism caused by these drugs on the insect GABA receptor was competitive. Flunitrazepam (10(-6) M) potentiated GABA responses, providing evidence for a benzodiazepine site on an insect GABA-receptor-chloride-channel complex.     

Aminopyridines block an inactivating potassium current having slow recovery kinetics.

The blocking action of aminopyridines on an inactivating K current (lKi) in GH3 pituitary cells was studied before and after altering the macroscopic decay of the current with N-bromoacetamide (NBA). The first depolarizing pulse delivered either seconds or minutes after beginning 4-aminopyridine (4AP) application, elicited a current with both a more rapid decay and a reduced peak amplitude. The rapid decay (or time-dependent block) was especially prominent in NBA-treated cells. With continued drug application, subsequent test pulses revealed a stable block of peak current, greater in NBA-treated than control cells. Recovery from block was enhanced by hyperpolarizing holding potentials and by the first depolarizing pulse delivered after prolonged recovery intervals. Unlike aminopyridine block of other K currents, there was no convincing evidence for voltage shifts in activation or inactivation, or for voltage and frequency-dependent unblock. Increasing the open probability of the channels did, however, facilitate the block. Although the behavior of currents in 4AP was suggestive of "open channel block," the block was not produced by 4-aminopyridine methiodide, a positively charged aminopyridine. Moreover, because partial block and recovery occurred without opening the channels we suggest that aminopyridines bind to, or near, this K channel, that this binding is enhanced by opening the channel, and that a conformational change is induced which mimics inactivation. Because recovery from block is enhanced by negative potentials, we suggest that aminopyridine molecules may become "trapped" by inactivation awaiting the slow process of reactivation to escape their binding sites.     

Effects of external protons on single cardiac sodium channels from guinea pig ventricular myocytes.

The effects of external protons on single sodium channel currents recorded from cell-attached patches on guinea pig ventricular myocytes were investigated. Extracellular protons reduce single channel current amplitude in a dose-dependent manner, consistent with a simple rapid channel block model where protons bind to a site within the channel with an apparent pKH of 5.10. The reduction in single channel current amplitude by protons is voltage independent between -70 and -20 mV. Increasing external proton concentration also shifts channel gating parameters to more positive voltages, consistent with previous macroscopic results. Similar voltage shifts are seen in the steady-state inactivation (h infinity) curve, the time constant for macroscopic current inactivation (tau h), and the first latency function describing channel activation. As pHo decreases from 7.4 to 5.5 the midpoint of the h infinity curve shifts from -107.6 +/- 2.6 mV (mean +/- SD, n = 16) to -94.3 +/- 1.9 mV (n = 3, P less than 0.001). These effects on channel gating are consistent with a reduction in negative surface potential due to titration of negative external surface charge. The Gouy-Chapman-Stern surface charge model incorporating specific proton binding provides an excellent fit to the dose-response curve for the shift in the midpoint of the h infinity curve with protons, yielding an estimate for total negative surface charge density of -1e/490 A2 and a pKH for proton binding of 5.16. By reducing external surface Na+ concentration, titration of negative surface charge can also quantitatively account for the reduction in single Na+ channel current amplitude, although we cannot rule out a potential role for channel block. Thus, titration by protons of a single class of negatively charged sites may account for effects on both single channel current amplitude and gating.     

Simple shifts in the voltage dependence of sodium channel gating caused by divalent cations

The effect of elevated divalent cation concentration on the kinetics of sodium ionic and gating currents was studied in voltage-clamped frog skeletal muscle fibers. Raising the Ca concentration from 2 to 40 mM resulted in nearly identical 30-mV shifts in the time courses of activation, inactivation, tail current decay, and ON and OFF gating currents, and in the steady state levels of inactivation, charge immobilization, and charge vs. voltage. Adding 38 mM Mg to the 2 mM Ca bathing a fiber produced a smaller shift of approximately 20 mV in gating current kinetics and the charge vs. voltage relationship. The results with both Ca and Mg are consistent with the hypothesis that elevated concentrations of these alkali earth cations alter Na channel gating by changing the membrane surface potential. The different shifts produced by Ca and Mg are consistent with the hypothesis that the two ions bind to fixed membrane surface charges with different affinities, in addition to possible screening.     

Modification of sodium and gating currents by amino group specific cross-linking and monofunctional reagents.

To test the possible role of lysine residues in Na channel function the effects of several imidoesters on Na and gating currents were studied in voltage-clamped single frog nerve fibers. Mono- and bisimidoesters were used. These reagents modify amino groups exclusively and do not change the net charge. The three bisimidoesters used easily introduce cross-links between neighboring amino groups. Their structure is almost identical; only the length of the spacers between the two amino-reactive groups is different. An irreversible reduction of Na currents and gating currents was observed with the longest (dimethyl suberimidate [DMS]) and the shortest (dimethyl adipimidate [DMA]) of the cross-linkers used. Of the three cross-linking reagents only the shortest made Na current inactivation slow and incomplete. The steady-state inactivation curve, h infinity (E), was shifted by greater than 25 mV in the hyperpolarizing direction by each of the reagents. The voltage dependence of activation, however, remained unchanged. Furthermore, the effects of two different monoimidoesters (ethyl acetimidate [EAI] and isethionyl acetimidate [IAI]) on gating currents were tested. EAI can penetrate a membrane, whereas IAI is membrane impermeant. IAI was almost without effect, whereas EAI caused a considerable reduction of the gating currents. EAI and DMS reduced the Qoff/Qon ratio without affecting the decay of the Na currents. The results show that lysine residues are critically involved in Na channel gating.     

Interaction between pentobarbital and GABA-activated ionic channels in rat cerebellar neurons

Using techniques of voltage clamping at the membrane, intracellular perfusion, and concentration clamping, GABA- and barbiturate-activated currents were investigated in single neurons isolated from the rat cerebellum. The dissociation constant for interaction between GABA and GABA receptors was measured at 3±0.8 × 10^–5 M. The presence of pentobarbital in the bathing solution exerts a potentiating effect on GABA-induced conductance in isolated neurons, shifting the dose-response curve for GABA towards lower concentration values without increasing peak chloride conductance. The concentrations at which GABA effects are potentiated range between 10^–6–10^–4 M. High concentrations of pentobarbital inhibit GABA-activated conductance; at concentrations in excess of 5 × 10^–4 M, it also brings about activation of chloride conductance, depressed by bicuculline and picrotoxin, in the absence of GABA. A short-term increase in membrane conductance is produced by rapid pentobarbital washout.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 22, No. 1, pp. 93–98, January–February, 1990.     

Quantal release of ATP in mouse cortex

Transient currents occur at rest in cortical neurones that reflect the quantal release of transmitters such as glutamate and gamma-aminobutyric acid (GABA). We found a bimodal amplitude distribution for spontaneously occurring inward currents recorded from mouse pyramidal neurones in situ, in acutely isolated brain slices superfused with picrotoxin. Larger events were blocked by glutamate receptor (AMPA, kainate) antagonists; smaller events were partially inhibited by P2X receptor antagonists suramin and PPADS. The decay of the larger events was selectively prolonged by cyclothiazide. Stimulation of single intracortical axons elicited quantal glutamate-mediated currents and also quantal currents with amplitudes corresponding to the smaller spontaneous inward currents. It is likely that the lower amplitude spontaneous events reflect packaged ATP release. This occurs with a lower probability than that of glutamate, and evokes unitary currents about half the amplitude of those mediated through AMPA receptors. Furthermore, the packets of ATP appear to be released from vesicle in a subset of glutamate-containing terminals.     

Studies of the role of gamma-aminobutyric acid in the hypothalamic control of feed intake in sheep

Fourteen sheep were used to study the role of gamma-aminobutyric acid (GABA) on the hypothalamic control of feed intake. Injections (1 microL) of pentobarbital (262 nmol) into preoptic and paraventricular areas induced feeding in satiated sheep. Injections of GABA into the same loci gave variable results, probably because the neuronal and glial uptake of GABA limits its effects. Muscimol, a GABA agonist with a higher affinity for postsynaptic GABA receptors than GABA, injected at doses from 0 to 0.750 nmol, gave a cubic dose-response curve; the highest feed intake was measured at 0.5 nmol. The response induced by muscimol was blocked by preinjections of two GABA antagonists, picrotoxin and bicuculline, with picrotoxin being more effective than bicuculline. Muscimol responsive loci were identified mainly in the preoptic, paraventricular, and anterior hypothalamus. The data suggests that neurons sensitive to gamma-aminobutyric acid may be implicated in the control of feed intake in sheep.     

Sodium channel inactivation in the crayfish giant axon. Must channels open before inactivating?

Experiments on sodium channel inactivation kinetics were performed on voltage-clamped crayfish giant axons. The primary goal was to investigate whether channels must open before inactivating. Voltage-clamp artifacts were minimized by the use of low-sodium solutions and full series resistance compensation, and the spatial uniformity of the currents was checked with a closely spaced pair of electrodes used to measure local current densities. For membrane potentials between -40 and +40 mV, sodium currents decay to zero with a single exponential time-course. The time constant for decay is a steep function of membrane potential. The time-course of inactivation measured with the double-pulse method is very similar to the decay of current at the same potential. Steady-state inactivation curves measured with different test pulses are identical. The time-course of double pulse inactivation shows a lag that roughly correlates with the opening of sodium channels, but detailed comparisons with the time course of the prepulse current suggest that it is not strictly necessary for channels to open before inactivating. Measurements of the potential dependence of the integral of sodium conductance area also inconsistent with the simplest cases of models in which channels must open before inactivating.