Inactivation of Macroscopic Late Na+ Current and Characteristics of Unitary Late Na+ Currents in Sensory Neurons

Mark D. Baker and Hugh Bostock

Sobell Department of Neurophysiology, Institute of Neurology, London WC1N 3BG, United Kingdom

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Baker, Mark D. and Hugh Bostock. Inactivation of macroscopic late Na+ current and characteristics of unitary late Na+ currents in sensory neurons. J. Neurophysiol. 80: 2538-2549, 1998. Na+ currents in adult rat large dorsal root ganglion neurons were recorded during long duration voltage-clamp steps by patch clamping whole cells and outside-out membrane patches. Na+ current present >60 ms after the onset of a depolarizing pulse (late Na+ current) underwent partial inactivation; it behaved as the sum of three kinetically distinct components, each of which was blocked by nanomolar concentrations of tetrodotoxin. Inactivation of one component (late-1) of the whole cell current reached equilibrium during the first 60 ms; repolarizing to -40 or -50 mV from potentials of -30 mV or more positive gave rise to a characteristic increase in current (tau  >=  5 ms), attributed to removal of inactivation. A second component (late-2) underwent slower inactivation (tau  > 80 ms) at potentials more positive than -80 mV, and steady-state inactivation appeared complete at -30 mV. In small membrane patches, bursts of brief openings (gamma  = 13-18 pS) were usually recorded. The distribution of burst durations indicated that two populations of channel were present with inactivation rates corresponding to late-1 and late-2 macroscopic currents. The persistent Na+ current in the whole cell that extended to potentials more positive than -30 mV appeared to correspond to sporadic, brief openings that were recorded in patches (mean open time ~0.1 ms) over a wide potential range. None of the three types of gating described corresponded to activation/inactivation gating overlap of fast transient currents.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

Many large dorsal root ganglion (DRG) neurons (>50 µm, apparent diameter) cultured from adult rats, generate a rapidly activating, sustained Na+ current, with an activation threshold ~15 mV more negative than that of the transient Na+ current (Baker and Bostock 1997a). At least part of this current persists over minutes and gives rise to a tetrodotoxin (TTX)-sensitive resting inward current over a wide range of membrane potentials. Because the persistent Na+ current activates at sufficiently negative potentials, it is partially activated at the normal resting potential. At nodes of Ranvier in human sensory axons, it amplifies local depolarizing currents, increasing and prolonging the resulting change in membrane potential (Bostock and Rothwell 1997). Schwindt and Crill (1995) provided evidence that the persistent Na+ current in neocortical pyramidal neurons also operates in this way, amplifying dendritic receptor operated currents. It therefore seems likely that persistent Na+ currents play an important role in determining membrane excitability in a variety of neurons.

The origin of the channel behavior generating persistent Na+ current in sensory neurons remains unresolved, although we previously concluded that activation-inactivation gating (mh) overlap of transient Na+ channels, operating alone, cannot explain the current (Baker and Bostock 1997a). In a study of unitary currents in membrane patches from frog skeletal muscle, Patlak and Ortiz (1986) reported that the gating characteristics of channels generating "late" Na+ currents differ from those predicted for mh overlap in several important respects, including exhibiting weak or absent activation voltage dependence and ultrashort open times. Because both transient and late currents were affected by slow inactivation (occurring over seconds), these authors concluded that the unitary late or persistent currents were generated by transient Na+ channels operating in unusual gating modes. More recently, this position was supported by the finding that single types of cloned brain and muscle sodium channel alpha -subunits can generate both transient and late current (Moorman et al. 1990; Ukomadu et al. 1992; Zhou et al. 1991). Evidence that an apparently normal, single, skeletal muscle Na+ channel can spontaneously enter a persistent gating mode was provided by Böhle and Benndorf (1995). Similarly, persistent Na+ current in cortical pyramidal cells was attributed to modal gating of transient channels, characterized by a possible spontaneous loss of fast inactivation (Alzheimer et al. 1993; Brown et al. 1994). Modal gating of transient Na+ channels (Patlak and Ortiz 1985) associated with late openings and slow macroscopic inactivation may therefore be a feature common to all transient channel types, including those in sensory neurons. Whether the gating repertoire of only one molecular channel type is sufficiently diverse to generate all the Na+ current in large sensory neurons is currently unclear. Blot hybridization and in situ hybridization suggests that homologs of rat brain Na+ channel I and/or II are involved in generating the Na+ currents in large DRG neurons in adults (Beckh 1990; Beckh et al. 1989; Waxman et al. 1994). Large neurons also express hNE/PN1 and NaCh6 (Black et al. 1996), the mouse homolog of the latter, Scn8a, being implicated in generating low-threshold and "resurgent" Na+ currents in Purkinje neurons (Raman et al. 1997).

This study had two aims, to test the hypothesis that the late Na+ current partially inactivates, consistent with the properties of steady-state Na+ currents previously reported from these neurons (Baker and Bostock 1997a) and to record the single-channel openings underlying late Na+ current. We present evidence that the late current is a composite, generated by populations of channels exhibiting diverse types of inactivation gating. Both the macroscopic inactivation kinetics and those associated with unitary currents reveal heterogeneous inactivation properties, none of which appear to correspond with the fast inactivation that terminates transient current.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Cell culture

Primary cultures of neurons were prepared from lumbar DRG of male Wistar rats (200-300 g), using a standard enzymatic dissociation procedure, as was fully described elsewhere (Baker and Bostock 1997a). After plating the dissociated cells onto poly-L-lysine (Sigma) coated glass cover slips in the culture wells of 12-well plates (Falcon), the cells were kept in a 37°C incubator with a 5% CO2 atmosphere for <= 3 days. In some experiments neurons were seeded on cover slips previously covered with a layer of Schwann cells and fibroblasts obtained from dissociated spinal roots. This modification conferred the advantage of improved neuron adhesion.

Electrophysiology

Coverslips with adherent neurons were mounted into a 35-mm diameter plastic petri dish, which formed the recording chamber. The recording solutions used were designed to eliminate all ionic currents, as far as possible, apart from Na+ currents. The extracellular solutions were similar to those described previously (Baker and Bostock 1997a), except we found it useful to include 1 mM K+ gluconate to reduce the residual outward rectification that resulted from incomplete dialysis of the intracellular contents. The normal extracellular solution contained the following (in mM) 135.6 Na-gluconate, 1 K-gluconate, 4.54 N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES) (Na), 5.46 HEPES, 1.1 Ca-gluconate2, 1.2 Mg-gluconate2, 5 4-aminopyridine (4-AP), 10 Cs-gluconate, and 10 tetraethylammonium (TEA) Cl. The pH was buffered to 7.2-7.3 (unless otherwise stated) with the addition of gluconic acid to neutralize the 4-AP. The normal internal solution contained (in mM) 143 CsCl, 3 ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (Na), 6.04 HEPES (Na), 3.96 HEPES, 1.21 CaCl2, 1.21 MgCl2, and 10 TEA Cl, pH 7.2-7.3. No attempt was made to block high-threshold voltage activated Ca2+ currents generated by these cells. Recordings were made with an Axopatch 200 amplifier (Axon Instruments) with CV202 headstage, and whole cell records were filtered with the four-pole Bessel filter on the amplifier; -3dB at 2 kHz. Membrane patch recordings were filtered at 5 kHz and sampled at 50 or 25 kHz. Control of command pulse protocols and data collection were carried out by either an IBM PC or a Dell PC running pClamp version 5 or 6 (Axon Instruments).

Whole cell recordings were made with fire-polished, thin wall glass electrodes (GC150TF-15 capillaries, Clark Electromedical; 1.5-mm OD, 1.17-mm ID). Once filled with recording solution, their initial resistances were usually between 1 and 3 MOmega . All electrodes were coated with Sylgard. The gluconate-Cl junction potential at the tip of the electrode was eliminated by applying a -10 mV offset to the holding potential (Baker and Bostock 1997a). All experiments on membrane patches utilized the outside-out configuration. Most patches were pulled with electrodes fabricated from standard thickness glass capillaries (GC150F-15; 1.5-mm OD, 0.86-mm ID) and fire polished, with an initial resistance between 8 and 20 MOmega . A few patches were pulled with thin-walled glass electrodes (~2 MOmega ), similar to those used for whole cell recording. In the former case, it seemed clear that the voltage dependence of the Na+ channels was shifted by ~20 mV in the hyperpolarizing direction, in comparison with that observed in the whole cell. Although noting that other investigators reported similar effects on the voltage dependence of ion channels in patches (e.g., Alzheimer et al. 1993; Fernandez et al. 1984) reasons for this were not investigated.

The whole cell capacity current transient generated on an imposed step in potential was routinely canceled with the simple resistance-capacitance (RC) circuit within the amplifier. The charging time-constant prediction facility of the Axopatch amplifier was always used, allowing rapid charging of the cell membrane capacity. In this way, the effective time constant for charging the membrane in response to an imposed step was reduced by <= 90%. Feedback series resistance (Rs) compensation was also always used and was set to >= 70%. The average cell capacitance, estimated from the potentiometer dial on the amplifier, was 120 pF (n = 18). The calculated voltage error caused by the residual, uncompensated Rs during the late currents was often <l mV and not >3 mV. No correction to the holding potential was made in subsequently derived current-membrane potential relations. The voltage error could be appreciable during large transient currents, as described for the data presented in Fig. 2A.


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FIG. 2. De-inactivation of late Na+ current. A: TTX-sensitive (100 nM) currents reveal de-inactivation of late Na+ current. Membrane potential held at -30 mV for 200 ms, then stepped more negative. De-inactivation evident as a conspicuous increase in the current amplitude recorded immediately on stepping to -40 (*) and -50 mV. Slow inactivation is also evident as slow decline in the current amplitude at -30 mV. Inset: peak TTX-sensitive transient current in same neuron evoked by command step to -30 mV = -11.4 nA, persistent current at 320-340 ms = -110.9 pA. B: plot of best-fit value for de-inactivation time constant against final membrane potential. Single exponential fitted over 20 ms from potential step to -40, -45, or -50 mV. Mean ± SE (4 neurons) plotted as solid circles. Data from 1 neuron plotted as open circle. C: maximal increase in current amplitude at -40 mV (late-1 component) is independent of amount of preceding slow inactivation at -30 mV. D: De-inactivating current amplitude at -40 mV expressed as a fraction of total current recorded during prepulse at 90-100 ms. Same neuron as in C.

Although we did not observe the growth of processes over the few days in which the large neurons survived in culture, a less than optimal voltage clamp in a few neurons resulted in inadequate control of large transient Na+ current. In such cells, the transient current repeatedly escaped the control of the clamp. Recordings from such neurons were discarded.

Voltage-clamp protocols

Whole cell late currents were commonly recorded with voltage-clamp steps from a negative prepulse potential (-110 mV) to a range of membrane potentials in 5- or 10-mV increments. Families of membrane currents were elicited by long duration clamp steps (e.g., 200 ms or 2 s). Whole cell leakage and residual capacity currents were removed by subtracting appropriately scaled currents evoked by reversed polarity clamp steps. During particularly stable experiments, it was possible to record sufficient data to subsequently compute "difference currents" from families of currents recorded before and after the superfusion of TTX (Sigma). The steady-state inactivation curve for the TTX-sensitive late current was obtained by using a 2-s prepulse to a range of potentials and then stepping the membrane potential to -20 mV, both in the presence and absence of TTX (50 nM). The current amplitude was measured at 60-70 ms, i.e., at a latency at which transient current made a minimal contribution to the total current. In membrane patches, leakage current and residual capacity transients generated in response to imposed potential steps were subtracted either by constructing records from traces without channel activity or by scaling responses to small, negative-going clamp steps.

Experiments were carried out at room temperature (20-25°C, unless otherwise stated). Solution changes were achieved by low-pressure local superfusion, driven by gravity.

Data analysis

Plots of the value of steady-state current availability (hinfinity ) for late Na+ current versus membrane potential (Em) were fitted with a Boltzmann relation of the form
<IT>h</IT><SUB>∞</SUB>= 1 − (<IT>A</IT>/{1 + exp[(<IT>E</IT><SUB>1/2</SUB>− <IT>E</IT><SUB>m</SUB>)/<IT>a</IT><SUB>h</SUB>]}) (1)
with a least-squares procedure (SigmaPlot, Jandel), where A is the maximum degree of inactivation, E1/2 is the membrane potential at which inactivation is half-maximal, and ah is the steepness parameter.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Inactivation of late Na+ current

When a series of increasing depolarizing voltage-clamp steps was applied to a DRG neuron, the first Na+ current to be activated showed no evidence of inactivation (Baker and Bostock 1997a) (Fig. 3A). With further depolarization the amplitude of the Na+ current increased greatly, and most of the current exhibited the fast inactivation characteristic of transient sodium current. Part of the late current failed to inactivate during long duration clamp steps lasting <= 2 s and thus appeared persistent (Fig. 1). The curve-fitting routines utilized always indicated that late current inactivation was partial, supporting previously obtained evidence that a part of this current is persistent. The decay of current after 20 ms at -30 mV was well described by the sum of two exponentials in addition to a persistent component, indicating that a part of the current inactivated with an intermediate time constant (tau  ~14 ms) and that a second part inactivated very slowly (tau  ~130 ms at -30 mV, Fig. 1). In this experiment an increase in leakage current during the application of TTX (measured at -110 mV) accounted for <30 pA of the steady-state current at -30 mV, estimated by the fitting procedure as -438.5 pA. The peak transient current at -30 mV exceeded -10 nA, saturating the recording. The relative amplitude of the transient and late currents at -30 mV could be more adequately determined for another neuron, recordings from which are shown in Fig. 2A. The residual, uncompensated series resistance error at the peak of the transient current was calculated as +8 mV. Assuming the transient current was fully activated at -30 mV, the current amplitude between 60 and 70 ms and 320 and 340 ms after the onset of the clamp step was 1.6 and 0.9% of the transient current, respectively. For convenience we use the term "late current" to describe the currents persisting after 60 ms, at which time the component with the intermediate time constant was small.


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FIG. 3. Characteristics of late Na+ current inactivation. A: family of TTX-sensitive (100 nM) currents, evoked by incrementing voltage-clamp steps (-80 to -30 mV) from a prepulse potential of -110 mV. Dotted line indicates a latency of 60 ms from the onset of clamp step. B: members of family of TTX-sensitive currents (50 nM) recorded during hinfinity protocol, with an incrementing prepulse 2 s in duration. Dotted lines indicate a latency of 60 ms and 780 ms. C: plot of slow time constant vs. membrane potential for TTX-sensitive current recorded from 2 neurons in 200 ms duration protocols. Smooth line given by the equation tau  = 1/[a exp(Em/b] + c, where a = 0.04 ms-1, b = 25.5 mV, and c = 63.2 ms. D: steady-state inactivation of late currents (hinfinity ), measured at 60-70 ms after 2 s prepulses to a range of potentials from -90 to -20 mV (pH 6.9-7.0 or 7.2-7.3). Values are means ± SE (n = 5) derived from TTX-sensitive difference currents (50 nM, 4 neurons) and residual inward current (1 neuron). Smooth curve is a Boltzmann relation drawn according to best-fit parameters (E1/2 = -56.2 mV, ah = 7.4 mV, A = 0.86). E: hinfinity relations for late-2 current, from current amplitude differences between latencies of 60-70 ms (T1) and 780-790 ms (T2); E1/2 = -56.2 mV, ah = 6.7 mV. Inset; plot of current amplitude values vs. membrane potential at T1 (closed circle) and T2 (open circle). Late-2 current may inactivate completely at -30 and -20 mV.


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FIG. 1. Tetrodotoxin (TTX)-sensitive transient and late current. A: transient and late Na+ currents, evoked by a clamp step from -110 mV to -30 mV, are blocked by superfusion of TTX. Control trace, 100 nM TTX, and wash. Horizontal dotted line indicates zero clamp current. B: TTX-sensitive currents derived by subtraction. Late Na+ current decays according to an intermediate and slow time constant (in this neuron the intermediate time constant was unusually prominent). Superimposed smooth curve is sum of 2 declining exponentials and a steady-state component, drawn according to best-fit parameters for data between 20 and 200 ms, inset; tau  = 14.3 ms (dotted curve 1) and 132.2 ms (dashed curve 2), with amplitudes of -896.7 and -461.43 at 20 ms, respectively. Steady-state component, -438.5 pA (solid line 3). Uncompensated voltage error during step to -30 mV caused by residual series resistance estimated as 3 mV at 20 ms.

Evidence suggesting that some of the late current had already undergone a faster inactivation was provided by protocols that allowed the partially inactivated current to de-inactivate (Fig. 2A). After inactivation occurred at -30 or 0 mV, stepping to more negative potentials caused not only rapid deactivation but also a conspicuous increase in current amplitude, attributable to the removal of inactivation. The de-inactivating currents were blocked, along with that inward current operating during the preceding clamp step, by nanomolar range TTX, confirming that they were Na+ currents. De-inactivation was difficult to perceive on stepping to potentials more negative than -50 mV. The time constant of de-inactivation was much shorter (~5 ms at -40 mV, Fig. 2B) than the slow inactivation observed during the preceding step. Because inactivation and de-inactivation are expected to have similar time courses at the same potential, this implies that two different types of inactivation were taking place.

Two types of late Na+ current distinguishable on the basis of inactivation kinetics

One explanation for the appearance of two distinct forms of late Na+ current inactivation is that the late current is usually a composite, each component being equally sensitive to nanomolar range TTX. We refer to the component of the late current undergoing intermediate rate de-inactivation as late-1, and that component undergoing slow inactivation as late-2. Evidence that late-1 current is not affected by the slow inactivation process (and that this must therefore be a characteristic of a second late current), is shown in Fig. 2C. The maximal increases in current amplitude after stepping to -40 mV were measured after holding the membrane potential at -30 mV for 100, 200, and 400 ms. Although slow inactivation was clearly more complete at 400 ms than at 100 ms, the amplitude of the de-inactivating current was not different and thus appeared independent of the degree of slow inactivation.

The kinetics of inactivation of the late-1 current in the whole cell are less certain than those of de-inactivation, although late-1 inactivation must have equilibrated within 60 ms of the start of an imposed clamp step. It is possible that the kinetics of the current at short latencies reflects inactivation of the late-1 component, assuming that no other Na+ current operates at the same time. It is also possible (if not likely) that inactivation of the late-1 plays a part in generating the second component of inactivation of transient nodal Na+ currents previously described (e.g., Benoit et al. 1985; Chiu 1977; Neumcke and Stämpfli 1982) and accounted for by subpopulations of channels exhibiting slower inactivation than those generating most of the transient current (e.g., Sigworth 1981). Although there remains some uncertainty about the origin of the intermediate component of inactivation in the whole cell, interpretation was simpler in recordings from small membrane patches. We found that ensemble mean currents could exhibit the kinetic characteristics appropriate for late-1 current in isolation (Fig. 4).


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FIG. 4. Na+ currents recorded in membrane patches. A: family of Na+ currents (average of 5 sequential recordings) from an outside-out patch pulled with a low resistance pipette. B: peak current-membrane potential relations (bullet ) for currents shown in A (left) and for normalized currents in patches from 4 neurons, plotted as means ± SE (right). SE usually smaller than the symbol size. Normalized peak current values for rat node of Ranvier (open circle ), calculated from previously published data (Neumke and Stampfli 1982). C: examples of ensemble mean currents (average of n = 99 records and n = 50 records, top and bottom traces respectively) recorded from outside-out patches pulled from 2 different neurons with higher resistance pipettes. Currents elicited on stepping from -100 to -50 mV. Imposed potential step functionally similar to that eliciting largest currents in A. Superimposed smooth curves drawn according to best-fit parameters between peak current and 80 ms. Top trace: sum of two declining exponentials tau  = 1.7 ms and 7.8 ms. Bottom trace, single declining exponential, tau  = 5.0 ms. In both cases the curve fitting procedure predicted incomplete inactivation.

Data on the late-2 component, obtained by two recording protocols, are shown in Fig. 3. TTX-sensitive difference currents were derived for the responses to 200 ms depolarizing voltage steps to show the time course of inactivation (Fig. 3A) and also for steps to -20 mV after 2 s duration, incrementing prepulses (Fig. 3 B), to assess the fraction of current remaining uninactivated (hinfinity protocol). In Fig. 3A, inactivation after 60 ms was imperceptible at -70 and -60 mV so that the late current appeared rectangular in shape. At more positive potentials inactivation became progressively evident, with a time constant that varied with membrane potential from ~80 ms to >200 ms (Fig. 3C). The availability (hinfinity ) of the total TTX-sensitive late current after a 2-s prepulse, recorded at 60-70 ms (shown as T1 in Fig. 3B), is illustrated in Fig. 3D. Inactivation was incomplete at -20 mV, in agreement with our previous report that some Na+ current persists at potentials as positive as 0 mV for several minutes (Baker and Bostock 1997a).

An hinfinity curve for the late-2 component alone may be derived by subtracting the current still remaining at 780-790 ms after the onset of the test step (T2) from the current at 60-70 ms (T1), Fig. 3E. There was no change in current amplitude between T1 and T2 after prepulses to -30 or -20 mV, consistent with the complete inactivation of the late-2 current at these potentials. The residual current at more positive potentials suggested the presence of a third current component, and this interpretation was supported by recordings of brief, sporadic channel openings in small patches, shown in Fig. 7. So far we were not able to separate the activation characteristics of the late current components, so we cannot say which are responsible for the steady-state current at potentials more negative than that at which transient Na+ current activates.


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FIG. 7. Brief, sporadic Na+ channel openings. A: example records from a small outside-out patch in which only brief, sporadic channel activity was evident. B: channel open time does not appear to depend on membrane potential. Open-time distribution at -80 + -85 mV and at -35 mV, solid bars and open bars, respectively (left); pooled measurements at potentials between -55 and -35 mV (right). Currents included if i >=  (5 pS × Em). Time constant of best single exponential fit to the data is 100 µs, implying about one-half of the actual number of events were lost to analysis. C: examples of openings at -60 mV, including a relatively long opening (~1 ms) that appears to include incompletely resolved brief closures. D: number of channel openings increases with depolarization, indicating channel activation was voltage dependent. Final Gaussian filter at 4 kHz.

Membrane patch recordings

In membrane patches pulled using low-resistance pipettes, activation of Na+ channels gave rise to transient currents with kinetics and voltage dependence similar to those recorded from nodes of Ranvier (Fig. 4). These recordings are consistent with the kinetic analysis of transient Na+ current by Kostyuk et al. (1981) in neurons from the same source, where inactivation was fast, occurring with a time constant of <2 ms at -40 mV and at room temperature. The behavior of Na+ channels in smaller patches differed from this in three respects. First, unitary currents appeared at potentials ~20 mV more negative than expected from whole cell current thresholds (see METHODS), and this was regarded as caused by a relative offset of the membrane potential. The offset not only affected the threshold for recruitment of noninactivating current (operating at the most negative potentials) but also the potential at which clear inactivation appeared (cf. Fig. 5). Second, allowing for effects of membrane potential, ensemble mean Na+ currents exhibited slower inactivation kinetics than those seen in large patches; third, inactivation kinetics varied from patch to patch. The kinetics of ensemble mean currents presented in Fig. 4C are consistent with the presence of channels generating fast transient current and late-1 current (top trace) and late-1 current without fast transient current (bottom trace). Although differences between the kinetics in the example currents may be partly due to uncertainties in the effective holding potential, these currents clearly inactivated more slowly than would be expected for fast transient current operating alone at any potential. Allowing for a membrane potential offset in our recordings, the late-1 inactivation kinetics appear similar to those reported by Caffrey et al. (1992) for fast Na+ current in excised patches from similar neurons.


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FIG. 5. Characteristics of unitary late Na+ currents. A: current-membrane potential relations for single-channel events recorded over 20 ms from onset of imposed clamp step for patches from 3 neurons at room temperature (left) and over 200 ms from 1 neuron at 15°C (right). Unconstrained regression lines give slope conductances of 17.5 and 16.7 pS, for plots on left and right, respectively. See text for details. B: recruitment of Na+ channels at near activation threshold in a small patch at 15°C. Traces on left were recorded on stepping to -85 mV (top) and -75 mV (bottom) from -110 mV. Right traces: ensemble mean records at the same potentials (average of n = 40 records and n = 45 records, top and bottom traces, respectively). C: examples of normal late Na+ channel openings at -75 mV.

Characteristics of unitary late Na+ currents

The unitary current-membrane potential relations for channels in four patches excised from different neurons are shown in Fig. 5A. In the left-hand plot, the data were obtained during voltage-clamp steps only 20 ms in duration. Mean values for the estimates of most common unitary current amplitudes are plotted for three patches and the slope conductance given by the unconstrained regression line fitted to the pooled data are 17.5 pS. Individual estimates of slope conductance lie within the range 13 to 18 pS. In the right-hand plot are data from one patch, where the voltage-clamp steps were 200 ms in duration and recording temperature was lowered to 15°C to reduce the rate of channel gating, allowing longer openings. For each potential, the number of individual estimates of the most common current amplitude varied from 5 to 29. The data are plotted as bars indicating ±1 SE, and the unconstrained regression line gives a slope conductance of 16.7 pS. Constraining the fit to pass through the theoretical reversal potential of +61 mV gave a chord conductance of 13.9 pS. These data show that there is no obvious difference between the conductance of channels opening early or late during a long voltage-clamp step, suggesting that Na+ channel conductance is the same throughout. In their study of late currents in cortical neurons, Alzheimer et al. (1993) reached a similar conclusion. The proposition that the conductance of Na+ channels generating transient and late current is the same is further supported by the results of nonstationary noise analysis in a different patch (see Fig. 6).


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FIG. 6. Fast transient current and late current generated by channels with the same conductance. A: superimposition of 52 records from a small outside-out patch. Na+ current evoked by stepping membrane potential from -100 to -50 mV. B: ensemble mean (negative going) and ensemble variance, sigma 2 (positive going), from the data in A. C: plot of sigma 2 against ensemble mean current for the first 80 ms from beginning of clamp step. Regression line has a slope of 1.67 pA (theoretical unitary current, i). D: amplitude histogram for single record exhibiting clear late openings (included in A) where N is number of sample points during a 200-ms clamp step. Sum of two Gaussian distributions superimposed on the data, according to best-fit parameters, indicating a unitary current of 1.61 pA for the late openings at -50 mV. Modal unitary current ~2 pA.

Examples of unitary current recordings and corresponding ensemble mean currents from a patch are seen in Fig. 5B. Typically, when first recruited at negative potentials, Na+ channels exhibited brief openings distributed throughout long-duration, voltage-clamp steps (-85 mV; Fig. 5B, top left trace). Although inactivation did not obviously affect the appearance of channel openings in many records, a partial macroscopic inactivation is evident in ensemble mean records (Fig. 5B, top right trace). A single declining exponential superimposed on the mean record shown is drawn with best-fit parameters and suggests that in this example the current declines to a steady-state amplitude 22% of its initial value, with a time constant of 25.2 ms. Greater depolarization recruits more channels and gives rise to a clear phase of inactivation (-75 mV; Fig. 5B, bottom traces), plainly evident on the ensemble mean trace (bottom right). The smooth curve superimposed on the ensemble mean data are drawn according to the sum of two exponentials, the major component declining with a time constant of 10.5 ms (initial amplitude -2.7 pA) and the minor component declining with a time constant of 39.2 ms (initial amplitude -1.2 pA), the extrapolated steady-state current being 1.5% of the initial current amplitude. Example single-channel currents are shown in Fig. 5C.

Use of ensemble variance

The derivation of ensemble variance is useful for two reasons. First, an estimate of the single channel conductance can be obtained throughout the response to a step change in membrane potential during both the transient current and the late current. Second, the ensemble variance can provide a robust method of demonstrating the presence of small, persistent currents. Assuming that the probability of any one channel being open is low, then the variance, sigma 2, about the mean current is equal to the mean current, I, multiplied by the unitary current, i (reviewed by Hille 1992), thus
σ<SUP>2</SUP>= <IT>Ii</IT> (2)
A plot of the ensemble variance against ensemble mean current for 52 traces during a step to -50 mV gives a linear relationship for a patch generating both transient and late Na+ current (Fig. 6). The slope of this relation gives the unitary current, and the single channel conductance, g, was estimated as a chord conductance, taking ENa to be +62 mV, where
<IT>g = i</IT>/(<IT>E</IT><SUB>m</SUB>− <IT>E</IT><SUB>Na</SUB>) (3)
The single channel conductance apparently appropriate for both transient and late current up to 80 ms after the onset of the clamp step was 14.9 pS, from a slope value of 1.67 pA. The linear relation indicates variance is directly proportional to mean current for either large or small values and suggests open probabilities must be low throughout the clamp step. For comparison, an amplitude histogram generated from a single sweep (included in the ensemble variance analysis), which clearly showed late openings, allowed a separate estimation of the single channel conductance by fitting Gaussian curves to the data (Fig. 6D). The chord conductance of the Na+ channel generating the late current was estimated to be 14.4 pS from the peak of the fitted curve. However, it is probable that the current amplitudes were not normally distributed. The most common amplitude indicated a slightly larger conductance (17.9 pS).

Multiple forms of inactivation gating behavior underlie late Na+ current

Na+ channels in membrane patches exhibited heterogeneous gating behaviors. One type of behavior involved voltage-dependent, apparently sporadic openings, with ultrashort open times (Fig. 7). This type of opening was commonly recorded throughout a wide membrane potential range and was reversibly blocked by 50 nM TTX. The channel open probability when gating in this manner was very low (<0.05). Although it was usual to observe similar behavior at the most negative potentials in every patch, it was recorded in isolation in only one patch. It corresponds well with the "drizzle" of brief Na+ channel events reported by Patlak and Ortiz (1986) for skeletal muscle and the late or background currents recorded by Mitrovic' et al. (1993) in rat axons (although the events recorded in muscle and axon membrane were not described as voltage dependent). Analysis of the currents recorded in this patch, measured if exceeding a 5 pS threshold, revealed that the number of events increased with depolarization but that the open time did not appear to depend on voltage. The mean open time, derived by fitting either one or the sum of two exponentials to the open-time histogram (weighted according to the y-value) was either 100 or 114 µs, respectively. As open times could only be resolved if they were >100 µs, this finding implies that about one-half of the total number of events were briefer than this and were lost to the analysis. The few longer duration events (~1 ms) may have been unresolved bursts of ultrashort openings (giving the currents a "flickery" appearance), such recordings suggesting the existence of a short-lived closed state (Fig. 7C). It was not possible to derive a convincing estimate of the normal conductance for a channel behaving in this way because the channels were not open long enough to allow discernment of a discrete current level and because of the effects of filtering on the event amplitudes. However, placement of horizontal cursors at the peaks of the largest events recorded between -75 and -35 mV not only indicated that many of them appeared close to the same amplitude but also allowed an estimate of the unitary slope conductance (17 pS).

A second type of behavior involved the production of short bursts of brief openings throughout long voltage-clamp steps or while the membrane potential was held constant for minutes (Fig. 8A). A third type was similar, except that burst durations could be >= 400 ms (Fig. 8B, top trace). Because at most potentials the channel openings were punctuated by closures, some of which were too short to be completely resolved, the burst openings had a flickery appearance. The bursting behavior was associated with a range of membrane potentials more positive than that at which sporadic activity was initially activated. Steps to sequentially more positive potentials above the threshold for initiating Na+ channel activity caused channel openings to appear to coalesce. At more positive potentials, where the degree of channel activation must have approached a maximum, the bursting behavior tended to disappear and be replaced by occasional, long boxlike openings (Fig. 8B), a change in gating similar to that recorded in cortical pyramidal neurons with depolarizations approaching 0 mV (Segal and Douglas 1997).


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FIG. 8. Patch records including putative late-1 and late-2 behavior. A: example of late-1 behavior, characterized by bursts of brief openings lasting up to a few tens of milliseconds (-55 mV, 15°C). B; example of less common putative late-2 behavior in the same patch as that in A, upper trace at -55 mV, lower trace at -35 mV. At -55 mV, long burst opening appears flickery, but at -35 mV flicker is less apparent, and opening becomes more boxlike. C: estimated burst duration histograms for data from same patch. Mean burst durations (tau ) from best single exponential fit to the data are 5.8 ms at -55 mV and 4.2 ms at -45 mV.

An analysis of late current channel burst durations, in recordings from a patch at -55 mV and at 15°C, indicated two clearly different populations of burst length, the shorter (and much more common) with a mean of 5.8 ms and the longer with a mean over an order of magnitude larger (Fig. 8B). In this patch long bursts, lasting for >100 ms, were present in <10 of 100 clamp steps to -55 mV, whereas the short bursts were usually present. The shorter burst duration seems to correspond well with the time constant associated with the de-inactivation of late-1 macroscopic current, the simplest hypothesis being that burst duration is determined by the rate of entry into the same inactivated state from which later escape (in the whole cell) gives rise to the late-1 current de-inactivation. The mean lengths for the shorter bursts in patches were clearly substantially longer than the open times necessary for channels generating most of the transient current (cf. Fig. 4C, top trace), by a factor of ~3, whereas the individual brief openings were too short to account for the kinetics of fast transient current. The separate population of longer burst lengths appear to be the events that underlie the late-2 component.

Unitary current de-inactivation

The late-1 current component in the whole cell is associated with a conspicuous increase in the total late current amplitude on stepping from a depolarized potential (e.g., -30 mV) to a more negative one, which we attribute to a de-inactivation. To confirm that the channels we recorded in patches can also give rise to this phenomenon, we looked for, and found, analogous unitary current behavior (Fig. 9). In the same patch as that shown in Fig. 9B, stepping the membrane potential from -40 to -60 mV gave rise to a delayed increase in the value of ensemble variance (calculated from 500 sequential sweeps), suggesting a slow reappearance from an inactivated state. In >= 80 sweeps, channel openings followed the potential steps that were arranged in short bursts (Fig. 9B), and the ensemble variance derived from these 80 records indicated a rise in average current taking place over 25 ms at room temperature. These results are consistent with the hypothesis that channels generating bursts of brief openings give rise to the increase in late current after a negative potential step, seen in whole cell recordings.


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FIG. 9. Unitary current de-inactivation. A: Na+ current recorded in small patch undergoes inactivation during a potential step to -50 mV. Membrane potential is then stepped 10 mV more negative, allowing de-inactivation of the current. Note change in sampling frequency from 1 to 50 kHz during the recording, temperature 15°C. B: de-inactivation in another patch, on stepping from -40 to -60 mV. Top two records: examples of single traces; bottom record: superposition of 5 similar traces. Channels operating during the step to -60 mV open in short bursts, and there is a clear tendency for their openings to occur with a delay after the clamp step; see text.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

We provided evidence that the late Na+ current in adult rat large sensory neurons undergoes a partial, voltage-dependent inactivation. This inactivation helps account for the differences in the current amplitudes recorded during voltage-clamp steps of a few hundreds of milliseconds and those measured after maintaining a range of holding potentials for >= 1.5 min (Baker and Bostock 1997a). We also recorded unitary currents in excised patches that correspond well with the late Na+ currents recorded in whole cell voltage clamp. Our findings reveal that those channels generating late and persistent current in peripheral nerve do not gate in the same way as the channels underlying most of the transient current. Furthermore, the recording of a unitary current correlate of a noninactivating macroscopic component of Na+ current helps to explain how steady-state Na+ current operates over such a wide membrane potential range.

Late Na+ current is generated by more than one population of Na+ channel, two of which can be discriminated on the basis of their inactivation kinetics (tau  ~5 ms and 100 ms for late-1 and late-2 at -30 mV, respectively). The late-2 component was usually the major component at 60 ms, but it may have inactivated completely at -30 mV (Fig. 3D). A slowly declining macroscopic late current, analogous to late-2, was reported previously in cardiac myocytes (Kunze et al. 1985). One hypothesis for the late-1 component is that it results from transient Na+ current activation-inactivation gating overlap (mh overlap) and therefore represents that persistent portion of the transient current predicted by a classical Hodgkin-Huxley analysis of macroscopic currents. We reject this hypothesis for three reasons. First, the time constant of de-inactivation of the late-1 component is too slow for it to be generated by the same channels as those underlying most of the transient current. The time constant for equilibration of fast inactivation in DRG neurons at -40 mV or more positive is <2 ms at 20°C (Kostyuk et al. 1981), approximately three times faster than late-1 inactivation at equivalent potentials. Second, many neurons exhibiting transient Na+ current do not appear to generate a significant late Na+ current of any kind (measured at the end of 300-ms duration protocols) (Baker and Bostock 1997a); third, this component is blocked by protons over the neutral pH range, whereas transient current is not (Baker and Bostock 1997b). However, the late-1 component may well equate with the minor, slow phase of Na+ current inactivation described by others at the node of Ranvier (e.g., Chiu 1977).

Single channel behavior underlying late current

The characteristics of unitary current recordings from small patches are compatible with our macroscopic recordings, but some aspects of the channel gating behavior could not have been predicted from the whole cell currents. We distinguished three typical patterns of openings: sporadic, brief openings, and short and long bursts, which could be responsible for a persistent current and the late-1 and late-2 inactivating components, respectively. Patlak and Ortiz (1986) reported similar brief openings and burst openings of Na+ channels in skeletal muscle membrane (however, they did not discriminate >1 type of burst opening). Mitrovic' et al. (1993) recorded sporadic, brief openings in sensory axon-attached patches (with open times of 150 and 120 µs at -30 and 0 mV, respectively), suggesting that such openings are a common feature of channel behavior in both sensory neurons and axons. Neither variance measurement nor unitary current measurement (Figs. 5 and 6) provided evidence that any of the late current components differed in conductance from each other or the transient channels. Long bursts, similar to those apparently underlying late-2 current, were reported previously. Patlak and Ortiz (1985) occasionally recorded ultralong Na+ channel bursts (150 ms in duration) in ventricular myocyte membrane patches in response to long voltage-clamp steps. More recently, it was reported that Na+ channels in cortical pyramidal neurons also give rise to similar long burst openings (Alzhiemer et al. 1993; Segal and Douglas 1997).

The presence of brief closures in our unitary current records (giving the currents a flickery appearance) may indicate the presence of a short-lived closed state. The replacement of late-2 bursting behavior with boxlike openings after sufficient depolarization could be explained by such a short-lived closed state becoming more unstable (i.e., the rate constant determining exit from the state would be voltage dependent and increase with depolarization). The voltage-dependent change from burst to boxlike openings is probably a reflection of the same process previously described as a lengthening of open times within a burst (Patlak and Ortiz 1986). All three types of channel opening appeared to flicker in a similar way, although this was only occasionally seen for the sporadic openings (Fig. 8C) because they were usually so brief. The principle difference among the three behaviors was the duration of the bursts, presumably related to the rate at which inactivation equilibrates. Paradoxically, equilibration with an inactivated state could be fast for those channels giving rise to the lowest threshold current, characterized by sporadic, brief openings. The sporadic, brief openings were recorded at the most negative potentials, where they may correspond to the low-threshold rectangular macroscopic current in Fig. 3A. They were also recorded at the most positive potentials, where they presumably contribute to the persistent current at 0 mV. Despite the wide potential range over which these openings were evident, the term background channel, used by Patlak and Ortiz (1986) to describe similar brief openings would not be appropriate because the sporadic openings exhibited clear voltage dependence (Fig. 7C). Each opening would thus represent a brief escape from inactivation (Patlak and Ortiz 1986), although not necessarily to a normal open state (Chandler and Meves 1970). This is an important possibility because selective removal or modification of that inactivation could dramatically alter the amplitude of late and persistent current over a strategic range of potentials, near rest, and provide a way of altering neuronal excitability.

Gating modes may underlie the late currents

The late currents we have characterized exhibited a low-threshold, an intermediate rate, and a slow rate of inactivation and a persistent component. Although it is well established that modal gating can affect the rate and completeness of Na+ channel inactivation, the same cannot be said for the apparent voltage threshold for activation. However, one recent preliminary report suggests that spontaneous entry of a Na+ channel into a slowly inactivating mode also shifts the voltage dependence of activation by >10 mV in the hyperpolarizing direction (Böhle et al. 1997). It is therefore possible that a single molecular type of Na+ channel could exhibit sufficiently diverse gating to explain all aspects of the late currents. However, given that large neurons contain several types of different Na+ channel mRNAs (Black et al. 1996) and that the low-threshold and late Na+ current we described appear similar in some respects to those generated by Scn8a in mice Purkinje neurons (Raman et al. 1997), it is possible that the late currents are generated by different Na+ channels than those generating most of the somatic transient Na+ current.

    ACKNOWLEDGEMENTS

  This work was supported by the Brain Research Trust, and a grant from the Medical Research Council.

    FOOTNOTES

  Address for reprint requests: M. D. Baker, Sobell Dept. of Neurophysiology, Institute of Neurology, Queen Square, London WC1N 3BG, United Kingdom.

  Received 17 March 1998; accepted in final form 28 July 1998.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/98 $5.00 Copyright ©1998 The American Physiological Society