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1 Department of Biological Sciences, Hopkins Marine Station of Stanford University, Pacific Grove, California 93950; 2 Department of Physiology and Biophysics, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801; and 3 Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305
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ABSTRACT |
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 Abstract
 Introduction
Methods
Results
Discussion
References
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Gilly, William F., Rhanor Gillette, and Matthew McFarlane. Fast and slow activation kinetics of voltage-gated sodium channels in molluscan neurons. J. Neurophysiol. 77: 2373-2384, 1997. Whole cell patch-clamp recordings of Na current (INa) were made under identical experimental conditions from isolated neurons from cephalopod (Loligo, Octopus) and gastropod (Aplysia, Pleurobranchaea, Doriopsilla) species to compare properties of activation gating. Voltage dependence of peak Na conductance (gNa) is very similar in all cases, but activation kinetics in the gastropod neurons studied are markedly slower. Kinetic differences are very pronounced only over the voltage range spanned by the gNa-voltage relation. At positive and negative extremes of voltage, activation and deactivation kinetics of INa are practically indistinguishable in all species studied. Voltage-dependent rate constants underlying activation of the slow type of Na channel found in gastropods thus appear to be much more voltage dependent than are the equivalent rates in the universally fast type of channel that predominates in cephalopods. Voltage dependence of inactivation kinetics shows a similar pattern and is representative of activation kinetics for the two types of Na channels. Neurons with fast Na channels can thus make much more rapid adjustments in the number of open Na channels at physiologically relevant voltages than would be possible with only slow Na channels. This capability appears to be an adaptation that is highly evolved in cephalopods, which are well known for their high-speed swimming behaviors. Similarities in slow and fast Na channel subtypes in molluscan and mammalian neurons are discussed.
Na channels appear to be universally responsible for action potential propagation along axons in metazoans and thus carry out high-speed transmission of information over relatively long distances. In line with this basic function, Na channels generally operate on a very fast time scale and show considerably less diversity of functional attributes than do voltage-gated calcium or potassium channels (Hille 1992 Neuronal somata without axons or large processes were isolated from enzyme-treated ganglia of Loligo opalescens (GFL of stellate ganglion), Octopus rubescens (stellate ganglion), Aplysia californica (pedal ganglion), Pleurobranchaea californica (pedal ganglion), and Doriopsilla albopunctata (cerebropleural ganglion), following procedures previously described, and manually dissociated cells were cultured at 15-17°C in an L-15-based medium (Gilly et al. 1990 Identification of INa in Aplysia neurons
TTX sensitivity is a property shared by many but not all Na channels (Anderson 1987
General features of INa in Aplysia and Loligo neurons
Figure 2A shows a family of prepulse-sensitive INa records obtained over a range of activating voltages from an Aplysia neuron. Figure 2B shows analogous records from a squid (Loligo) GFL neuron under the same recording conditions. General features of INa are similar in both species. Inactivating INa first turns on at around
Differential voltage dependence of activation kinetics in Aplysia versus Loligo
Aplysia INa in Fig. 2 activates more slowly than does INa in squid at every voltage. Although INa activation kinetics in each species become progressively faster with increasing depolarization, the degree to which activation kinetics change with voltage differs. This differential voltage dependence is revealed by comparison of Aplysia and Loligo INa traces (scaled to the same peak value) obtained at a very positive voltage (+60 mV, Fig. 4A) with records obtained at an intermediate voltage (
Slow- and fast-type INa in cephalopod and gastropod neurons
Similar experiments were carried out on neurons isolated from other gastropod and cephalopod species. Fast activation kinetics at all voltages as seen in Loligo (Fig. 6A) are also typically observed in Octopus (Fig. 6B). On the other hand, INa kinetic characteristics of Aplysia (Fig. 6C) are shared by the gastropods Pleurobranchaea (Fig. 6D) and Doriopsilla (Fig. 6E). From these data, it is obvious that the gastropod neurons tend to exhibit slower kinetics, and the most prominent differences are observed for negative voltages (e.g.,
Inactivation kinetics follow activation kinetics in all species
Coupling of activation gating to inactivation in Na channels is well established in squid giant axon (Armstrong 1981
Zn slows activation kinetics of both Loligo and Aplysia INa
Differences in INa activation kinetics in cephalopods and gastropods as described above resemble the effects of Zn ions on INa in squid axon (Gilly and Armstrong 1982
Results described in this paper indicate that INa in randomly selected small gastropod neurons shows substantially slower activation kinetics than that in squid GFL cells or their giant axons. This general conclusion has been reached in previous studies of giant neurons of several gastropod species (Adams and Gage 1979a Functional significance of Na channel kinetic differences in molluscan neurons
These results are significant from both a biological and biophysical viewpoint. In effect, the shallow voltage dependence of activation kinetics shown by the fast type of Na channel in squid GFL cells means that these channels are fast at all voltages. This would permit very rapid adjustments in the number of open channels over the entire activation voltage range, including near threshold for action potential generation. Such rapid responses would not be possible with the slow-type channel described here in the gastropod neurons studied, because at the highly positive and negative voltages at which kinetics become fast, the channels would essentially either be all open or all closed, respectively.
Comparison of slow INa in mollusks and mammals
Studies of mammalian Na channels have also revealed the existence of slowly activating channel types. In particular, results described in this paper show some similarity to data obtained with patch-clamp methods on INa in rat neurons isolated from dorsal root ganglia (DRG). DRG neurons show two distinct kinetic types of INa in whole cell recordings. One type, primarily associated with small neurons, is very resistant to TTX and displays activation kinetics that are up to 5 times slower than the "normal" TTX-sensitive INa typically found in larger neurons (Elliott and Elliott 1993 Biophysical and molecular basis of the slow type of INa
Large kinetic differences between the two types of INa in molluscan neurons are restricted to the intermediate voltage range spanned by the gNa-V curve. In this range, both forward and backward rate constants underlying channel gating are significant, with the forward rates being rate limiting for activation (Armstrong and Gilly 1979
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
). Several important differences in Na channel functional properties have emerged, however, as patch-clamp methods have greatly expanded the range of cell types and species from which voltage-clamp data can be obtained. Many studies have focused on toxin sensitivity, especially to tetrodotoxin (TTX) and saxitoxin, and on inactivation properties, because these characteristics can change dramatically in mammalian nerve and muscle cells during normal development or after injury, and mechanisms underlying these functional differences have been successfully approached with molecular structure-function analysis (Catterall 1994). Moreover, several human genetic diseases have been identified that are due to defects in Na channel 
-subunits that affect inactivation (Catterall 1992; Ji et al. 1995; Wang et al. 1995).
; Sigworth 1993; Yang et al. 1996) are highly conserved between phylogenetically distant organisms such as jellyfish (Anderson et al. 1993), squid (Rosenthal and Gilly 1993), flies (Salkoff et al. 1987), and vertebrates (Noda et al. 1984). Activation kinetics, however, are somewhat more variable, and Na current (INa) with unusually slow activation kinetics has been reported in mammalian neurons (Elliott and Elliott 1993; Kostyuk et al. 1981; Ogata and Tatebayashi 1993; Roy and Narahashi 1992) and glial cells (Barres et al. 1989). Neither the functional significance nor the molecular basis of slow activation kinetics has yet been clarified (Hoehn et al. 1993), but slow Na channels are probably more important to integration and pacemaking (Yoshitaka 1996) in neurons than to axonal transmission (Llinas 1988).
), including terrestrial (Helix: Kostyuk et al. 1977), aquatic (Lymnaea: Kostyuk et al. 1977), and marine (Aplysia: Adams and Gage 1979a,b; Byrne 1980) species. In all cases, gastropod INa appeared to activate and inactivate substantially more slowly than INa in the giant axon of squid, a cephalopod mollusk. Among the gastropod studies, the most complete analysis was that of Adams and Gage (1979a,b), performed in R15 of the abdominal ganglion of Aplysia juliana. Although it is clear that Aplysia INa activates slowly, quantitative comparison with data from squid axon or other fast Na channel types has not been carried out. Deactivation (closing) kinetics, for example, have not reported.
).
; Lucero et al. 1992), and the fast versus slow distinctions probably represent functional adaptations that have evolved to different degrees in the species studied here, which show extreme differences in speed of behavioral acts, such as locomotion.
METHODS
Abstract
Introduction
Methods
Results
Discussion
References
). Squid GFL neurons require several days in culture before sizable INa develops (Brismar and Gilly 1987; Gilly et al. 1990), and recordings from this cell type were therefore carried out after 3-6 days in vitro. Neurons from other species already displayed reasonable INa on the day of isolation, and most recordings from these species were performed after 1 (for gastropods) to 3 (for Octopus) days in vitro.
feedback resistor in the headstage (Gilly and Brismar 1989). Both soft (001, KG-12) and hard (7052) glasses were used. Although it is more difficult to attain a suitable taper for whole cell recordings with the hard glass, the series resistance tends to be more stable over time, at least with GFL neurons. Electrodes were generally of 0.3-0.5 M resistance when filled with internal solution (see below). Series resistance and input capacitance compensation were optimally adjusted throughout the course of each experiment by evaluating the current transient accompanying a 
10-mV voltage step. These currents were used to determine input resistance and capacitance, cell charging time constant (typically 30-60 µs), and the effective series resistance (0.4-0.8 M) as described elsewhere (Armstrong and Gilly 1992). Clamp performance was identical for neurons in all species studied.
4) in which hyperpolarizing control pulses were one fourth the amplitude of the test pulse and were delivered from the holding potential of 80 mV. Peak INa was converted to gNa with the use of the relationship gNa = INa/(V VNa), where V is pipette voltage and VNa is reversal potential.
-2-ethanesulfonic acid (HEPES), pH 7.8. The internal solution contained (in mM) 100 sodium glutamate, 50 NaF, 50 NaCl, 300 tetramethyl ammonium glutamate, 10 Na2 ethylene glycol-bis(
-aminoethyl ether)-N,N,N,N-tetraacetic acid, 25 tetraethylammonium chloride, and 10 HEPES, pH 7.8. Test and control solutions for experiments with ZnCl2 were carefully adjusted to pH 7.0-7.1.
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
; Barchi 1987; Narahashi 1974). Neurons of gastropod mollusks are generally extremely resistant to TTX (Adams et al. 1980), but Aplysia displays Na channels that are moderately TTX sensitive (Geduldig and Greuner 1970). This sensitivity permits the use of this highly specific toxin for isolating INa in whole cell recordings. Figure 1A shows current at 0 mV recorded in the absence and presence of 100 nM TTX, a concentration that blocks approximately half of the transient inward current and the fast portion of the tail current. Subtraction of these traces defines TTX-sensitive INa (Fig. 1B).
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FIG. 1.
Na current (INa) in an Aplysia neuron revealed by tetrodotoxin (TTX) and prepulse subtraction. A: currents recorded in the absence and presence (+TTX) of 200 nM TTX during a 5-ms step to 0 mV from a holding potential of 80 mV. B: subtraction of traces in A yields TTX-sensitive INa. C: currents recorded at 0 mV without and with (+pre) a 100-ms prepulse to 20 mV. D: subtraction of traces in C yields prepulse-sensitive INa. E: TTX- and prepulse-sensitive INa compared by scaling traces from B and D to the same peak value during the voltage step.
20 mV to inactivate INa. The transient inward current during the pulse and the fast component of the tail current are eliminated by the prepulse, and subtraction of these records yields the prepulse-sensitive INa trace illustrated in Fig. 1D. In this case, some of the slow component of the tail current, which is most likely carried by calcium or potassium channels, is also reduced by the prepulse.
30 mV and reverses between +20 and +30 mV. Aplysia INa activates and inactivates more slowly than does INa in squid at every voltage. INa-voltage and gNa-voltage (gNa-V) curves from these neurons are compared in Fig. 2C after normalization to maximal INa (1.0) or gNa (+1.0), respectively. Voltage dependence of gNa in Aplysia and Loligo thus appears to be indistinguishable.
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FIG. 2.
Comparison of prepulse-sensitive INa in Aplysia and Loligo. A: 5-ms voltage steps were delivered in an Aplysia neuron with and without a prepulse to 20 mV for 100 ms. Prepulse-sensitive INa, obtained by subtraction of these records, is illustrated at the indicated test voltages. B: family of prepulse-sensitive INa for a Loligo giant fiber lobe (GFL) neuron under the same experimental conditions. C: peak values of prepulse-sensitive INa during 5-ms voltage steps from Aplysia (
) and Loligo (
) neurons plotted against pipette voltage. Both INa-voltage (INa-V) relations have been normalized to a value of 1.0 at 0 mV. Na conductance (gNa) was determined (see METHODS) for the same 5-ms pulses in Aplysia (
) and Loligo (
) and is plotted against voltage after normalization to a value of +1.0 at +40 mV.
; Stimers et al. 1985) has minimal effects on activation kinetics, and it is thus likely that activation kinetics can be adequately assessed in GFL cells with intact inactivation.
; Wang et al. 1985). Activation kinetics of INa in GFL neurons are also largely unaltered at either negative (Fig. 3C) or positive (Fig. 3D) voltages by chloramine-T treatment.
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FIG. 3.
Activation kinetics of squid INa are not greatly affected by removal of inactivation. A: currents were recorded at the indicated voltages in a GFL neuron. These records have not been prepulse subtracted, and steady current at the end of each pulse is primarily noninactivating INa (Gilly and Brismar 1989 ). B: currents in the same neuron were recorded after bath application of 0.75 mM chloramine-T and subsequent washout. The fraction of inactivating current is greatly reduced. C: comparison of the records at 10 mV obtained before and after (*) chloramine-T treatment after scaling to the same peak amplitude. D: comparison of records as in C made at +60 mV.
10 mV, Fig. 4B). It is visually obvious that the discrepancy in activation kinetics is greater at 10 mV than at +60 mV. At negative voltages, deactivation (closing) kinetics of INa are comparable in both species, as evidenced by tail currents recorded after strong, brief activating pulses (measured at 80 mV, Fig. 4C). Aplysia and Loligo Na channels thus close with the same time course at a very negative voltages and open with similar rates at a very positive voltage. Rates controlling channel opening in the intermediate voltage range (near 0 mV) appear to be very different, however.
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FIG. 4.
Kinetic comparison of Aplysia and Loligo INa. A: examples of prepulse-sensitive INa for 5-ms pulses to +60 mV have been scaled to the same peak value to compare kinetics. B: examples of INa recorded at 10 mV are scaled to the same peak. INa is much slower in Aplysia than in Loligo. C: tail currents, recorded at 60 mV after a 0.4-ms pulse to +60 mV, are scaled to the same peak. No difference exists in tail current kinetics in the 2 species.
OFF), obtained from fitting a single exponential to tail currents, converge on a time constant of ~50 µs, and tails recorded negative to 50 mV are essentially indistinguishable [Fig. 5A; (Aplysia) vs. (Loligo)]. A time constant characterizing activation (ON) was derived by fitting a single exponential to the final approach (~25%) to peak INa, and this parameter is approximately equivalent to m of Hodgkin and Huxley (1952). ON values in both species [
(Aplysia) vs. 
(Loligo)] also converge at very positive voltages and become progressively slower at more negative voltages. The apparent voltage dependence of ON, however, is much greater in Aplysia than in Loligo. A similar difference between Aplysia and Loligo exists for the half time to peak INa (t1/2, Fig. 5B), a parameter that is a sensitive indicator of the delay in INa activation. Thus Aplysia Na channels operate much more slowly than do those in Loligo over the intermediate voltage range spanned by the gNa-V curve (i.e., 20 to +20 mV). At positive and negative extremes of voltage, Na channels of each species are kinetically similar.
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FIG. 5.
Voltage dependence of INa activation ( ON) and deactivation (OFF) parameters. A: values for OFF were determined for Aplysia () and Loligo () by fitting single exponentials to tail currents at various potentials after an 0.4-ms activating pulse to +60 mV. ON values were calculated by fitting a single exponential to the final activating portion of prepulse-sensitive INa, for Aplysia () and Loligo (). B: half time to peak INa (t1/2) values for INa turn-on is plotted as a function of activation potential for Aplysia () and Loligo ().
10 mV, indicated by *), where they are clearly manifested as a large difference in t1/2 values for activation (marked with arrowheads for each 10 mV trace).
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FIG. 6.
Fast INa of cephalopods vs. slow INa of gastropods. A-E: families of prepulse-sensitive INa for Loligo (A), Octopus (B), Aplysia (C), Pleurobranchaea (D), and Doriopsilla (E). Currents from each species were obtained with the use of the same solutions and experimental conditions. In each plot, the INa trace at 10 mV is indicated (*), and the location of the half time for 10-mV pulses is marked with an arrowhead. t1/2 values: A, 0.99 ms; B, 1.28 ms; C, 1.92 ms; D, 2.57 ms; E, 2.72 ms.
OFF, Fig. 7B) in an assortment of neurons from these five species reinforces the differences described above between Aplysia and Loligo (Figs. 4 and 5). The relationship between t1/2 and voltage in the cephalopod cells (, ) is much less steep than that in the gastropod neurons (, , ). Differences in the voltage dependence of OFF among these examples are much less marked (Fig. 7B), and values at subthreshold voltages (i.e., negative to 50 mV) are basically indistinguishable. Peak gNa-V relationships are also quite comparable in the gastropod and cephalopod neurons (Fig. 7C). In all cases, the intermediate voltage range spanned by the gNa-V curve is the same region where the differential voltage dependence of activation kinetics is most prominent.
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FIG. 7.
Comparison of activation and deactivation properties in cephalopod and gastropod species. Data in A and B are plotted from a number (n) of individual neurons from each species with the use of the same symbol type (i.e., data are not mean values). A: values for t1/2 for prepulse-sensitive INa are plotted as a function of activation voltage for Loligo ( ; n = 4), Octopus (; n = 6), Aplysia (; n = 3), Pleurobranchaea (; n = 2), and Doriopsilla (, n = 3). B: OFF values obtained from tail currents displayed in an analogous manner (Loligo, n = 3; Octopus, n = 1; Aplysia, n = 3; Pleurobranchaea, n = 2; Doriopsilla, n = 2). C: normalized (maximal gNa = 1) gNa-voltage (gNa-V) relations derived from prepulse-sensitive INa for the 5 neurons included in Fig. 6. Symbol types for each species are the same as in A and B. Dashed curve: single Boltzmann function (k = 9.4 mV, V1/2 = 2.2 mV) fitted to values obtained from Loligo, where gNa = 1/{1 + exp[(V1/2 V)/k]} and V is voltage.
; Lucero et al. 1992). Fast-type INa was never encountered in a gastropod neuron in this study, although we by no means carried out an exhaustive survery (see also DISCUSSION).
; Armstrong and Bezanilla 1977) as well as in mammalian preparations (Aldrich et al. 1983; Cota and Armstrong 1989). Coupling is also apparent in every species studied here, although the degree (and undoubtedly mechanism) of coupling may not be identical in each case. At voltages where peak INa was large enough to measure accurately, a single exponetial was fit to the falling phase of INa, and the time constant thus obtained is defined as the inactivation time constant (INACT). At voltages where little or no INa flowed, prepulses of varying duration were applied, and the decay time constant of INa (test voltage of +60 mV) was determined from a semilog plot to yield INACT. Results from cephalopod and gastropod neurons are compared in Fig. 8A. INACT of the gastropod Na channels exhibits a much more marked voltage dependence in the intermediate voltage range than it does in the cephalopod Na channels studied, and INACT values in both groups converge at the positive voltage extreme. This is analogous to the pattern shown by activation kinetic parameters.
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FIG. 8.
Inactivation kinetics follow activation kinetics in cephalopod and gastropod neurons. A: time constants for inactivation ( INACT) for the 5 species (symbols as in Fig. 7) plotted against activating pulse voltage (see text for measurement details). B: voltage dependence of "steady-state" INa inactivation was determined by measuring the peak residual INa during a 5-ms test pulse to +140 mV after a 100-ms prepulse to various potentials. Values for half-inactivation can be estimated from the illustrated fits with single Boltzmann functions (Loligo, 52.5 mV; Octopus, 27.6 mV; Aplysia, 36.5 mV; Pleurobranchaea, 23.7 mV; Doriopsilla, 25.3 mV).
of Hodgkin and Huxley 1952) is basically the same in each case, the position on the voltage axis for the squid data are displaced in the negative direction compared with data from the other species, including Octopus, which shows fast-type INa. We have not yet carefully studied inactivation kinetics in any species to quantitatively account for the specific contributions of "fast" and "slow" inactivation processes, and differences in this regard between these species may underlie the large dispersion of steady-state inactivation curves relative to the close match of the gNa-V curves.
). In the latter case, activation kinetics are significantly slowed, deactivation kinetics are essentially unchanged, and the gNa-V curve is slightly shifted in the depolarizing direction. Figure 9 compares the effects of 20 mM ZnCl2 on INa parameters from Loligo and Aplysia. In both cases, results similar to those described above for squid axon are observed for the voltage dependence oft1/2 (Fig. 9, A and D), gNa (Fig. 9, B and E), and OFF (Fig. 9, C and F). Similar results were obtained on a Pleurobranchaea neuron (data not shown). Zn thus perturbs activation gating of both Na channel types in a similar manner.
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FIG. 9.
Zn slows INa activation kinetics in both Loligo and Aplysia. A-C: effects of Zn on the voltage dependence oft1/2 (A), gNa (B), and OFF (C) in a Loligo neuron. Data were obtained in the absence () and presence () of 20 mM ZnCl2. gNa-V curve is fit with a single Boltzmann function for control (
; k = 5.6 mV, V1/2 = 13.3 mV) and Zn-containing solutions (- - -; k = 6.0 mV, V1/2 = 7.5 mV). Both control and Zn solutions contained 20 mM CaCl2. D-F: effect of Zn on the voltage dependence of t1/2 (D), gNa (E), and OFF (F) in an Aplysia neuron. Data were obtained in the absence () and presence () of 20 mM ZnCl2. gNa-V curve is fit with single Boltzmann function for control (; k = 7.5 mV, V1/2 = 7.3 mV) and Zn-containing solutions (- - -; k = 8.1 mV, V1/2 = +1.8 mV).
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
,b; Byrne 1980; Kostyuk et al. 1977; Neher 1971). Quantitative comparisons in the present work, however, require that this conclusion be qualified in an important waythe apparent kinetic differences depend on the voltage at which the comparison is made. For a strong depolarization, Na channel activation kinetics are reasonably comparable in the gastropod neurons and squid GFL cells (or giant axon), but for small depolarizations, kinetics in GFL neurons are much faster. Similarly, deactivation kinetics measured from tail currents are also comparable at very negative voltages. Thus it is only over the intermediate voltage range, i.e., that spanned by the gNa-V curve, that the slowness of gastropod Na channels is evident.
). Na channels expressed in the somata of GFL neurons have activation properties identical to those of axonal Na channels and appear to represent an ectopic population of axonal channels seen only after several days in vitro (Gilly et al. 1990, 1995).
).
). If such channels were of general importance, their presence might be expected in cephalopods as well as gastropods. In support of this idea, INa in Loligo with a kinetic pattern much like that shown here for gastropod INa has been reported in small, presumptive interneurons of the stellate ganglion (Liu and Gilly 1995) and in primary sensory neurons that show spontaneous, burst-type repetitive firing (Lucero et al. 1992). Slowly activating Na channels are thus likely to reflect important functional specializations in different molluscan neurons rather than a general difference between gastropod and cephalopod taxa.
), whereas Na channels appear to be absent in Aplysia muscle fibers (Brezina et al. 1994).
; Susswein et al. 1984), however, are relatively slow compared with the capabilties of Loligo and most cephalopods. Examination of other molluscan classes that show very rapid movements, such as the pteropods (Hermans and Satterlie 1992), may also reveal fast-type Na channels. Another approach would be to investigate INa in Nautilus because of the slow movements and evolutionary position of this cephalopod. During the course of this study we made some preliminary recordings form isolated Nautilus neurons, and INa over the intermediate voltage range appeared to be considerably faster than that in the gastropods studied in this paper. If this is the case, fast-type Na channels may have evolved very early in the evolutionary history of cephalopods, before the appearance of nonshelled forms that may have displayed rapid behaviors characteristic of extant species.
; Ogata and Tatebayashi 1993; Roy and Narahashi 1992). Steepness of the gNa-V relation of both types is similar, but the curve for slow, TTX-resistant channels is shifted significantly (+10 to +25 mV) relative to that for fast, TTX-sensitive gNa. Voltage dependence of inactivation shows a larger difference between the two INa types (+25 to +40 mV shift), and disagreement exists over whether the steepness is different in the two cases. Such comparisons must be carried out with caution, however, because the relevant DRG studies were carried out over a significant temperature range, and mammalian Na channels in both muscle cells (Kirsch and Sykes 1987) and neurons (Sah et al. 1988) can show anomalies in gating in this range.
; Lucero et al. 1992). Finally, in DRG neurons, activation and deactivation kinetics of the TTX-resistant INa are slower at all voltages, including the positive and negative extremes at which kinetic parameters for the two molluscan INa types converge.
). Voltage dependence of these forward rate constants thus appears to be significantly less steep in the universally fast Na channel type found in squid GFL neurons and giant axons. At present, the biophysical basis for this difference is unknown, but further analysis of ionic and gating current kinetics (Armstrong 1981, 1992) for these Na channel types may yield insights into the physical mechanisms that determine the rates at which voltage-gated channels can adjust their position in response to changes in voltage (Stevens 1978).
; Stuhmer et al. 1989; Yang et al. 1996). Recently a cDNA encoding a Na channel -subunit (PN3) was identified that is primarily expressed in small DRG neurons in the rat (Sangameswaran et al. 1996). Functional expression of PN3 in Xenopus oocytes resulted in TTX-resistant INa, but activation kinetics were not unusually slow. If the PN3 channel corresponds to the TTX-resistant, slowly activating Na channel type in DRG neurons discussed above, factors other than the primary structure may be critical to determining activation kinetics of this channel type.
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ACKNOWLEDGEMENTS |
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We thank Dr. Stuart H. Thompson for comments on the manuscript. We are grateful to the providers of living specimens: M. Morris, Sea Life Supply, Sand City, CA for Aplysia, Pleurobranchaea, and Octopus; the Monterey fishing community for Loligo; and the National Cephalopod Resource Center, UTMB, Galveston, TX for Nautilus.
This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-17510 to W. F. Gilly and a Ford Foundation fellowship to M. MacFarlane.
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FOOTNOTES |
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Address for reprint requests: W. F. Gilly, Hopkins Marine Station, Pacific Grove, CA 93950.
Received 24 September 1996; accepted in final form 19 December 1996.
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REFERENCES |
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Abstract
Introduction
Methods
Results
Discussion
References
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an approach to studying nerve excitability.
Biophys. J.
22: 295-306, 1978.[Medline]steady state properties.
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