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1 Department of Physiology and Pharmacology, State University of New York, Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, NY 11203, USA
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(Received 26 July 2006;
accepted after revision 16 October 2006; first published online 19 October 2006)
Corresponding author M. Vassalle: Department of Physiology, Box 31, SUNY, Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, NY 11203, USA. Email: mario.vassalle{at}downstate.edu
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Introduction |
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Since TTX selectively blocks Na+ channels (Naharashi, 1974), the shortening of the plateau with no or smaller effects on the rate of rise of the upstroke suggests a sodium current different from the fast-activating and -inactivating INa (here labelled INa1). The TTX-sensitive sodium current in question could be one or more of the following currents that have been described in cardiac tissues: (i) the background sodium current (Coraboeuf et al. 1979; Zilberter et al. 1994); (ii) the window current (Attwell et al. 1979); (iii) the persistent sodium current found in rat ventricular myocytes (Saint et al. 1992); and (iv) a slowly inactivating current that has been described in canine (Gintant et al. 1984) and rabbit (Carmeliet, 1987) Purkinje fibres. However, it should be noted at the outset that the AP duration of ventricular myocytes is very little affected by TTX (Coraboeuf et al. 1979; Bhattacharyya & Vassalle, 1982), by local anaesthetics (Vassalle & Bhattacharyya, 1980) or by veratridine (Iacono & Vassalle, 1990).
The general aim of the present experiments was to investigate, by means of patch voltage-clamp techniques, whether a slowly inactivating sodium component is responsible for the marked shortening of the Purkinje fibre plateau induced by TTX. In fact, a slowly inactivating inward current was found at less negative potentials. Therefore, we addressed the question of whether the slowly inactivating component (tentatively labelled here INa2) resulted from the slow inactivation of a sodium channel and whether INa2 had characteristics different from those of INa1. We used single Purkinje cells in order to avoid the complications related to ion depletion and accumulation in narrow extracellular spaces (Baumgarten & Isenberg, 1977; Cohen & Kline, 1982). In addition, the use of single cells affords a better control of membrane potential during voltage-clamp depolarizing steps.
The specific aims were to determine some of the characteristics of INa2 (threshold, voltage dependence and time constants of inactivation), as well as its behaviour during depolarizing and repolarizing ramps with different holding potentials and different slopes Also, several ion channel blockers (tetrodotoxin, lignocaine, cadmium and manganese) were tested to verify the identity of the ion involved in INa2.
The results obtained indicate that INa2 is a slowly inactivating Na+ current which has characteristics (and functions) different from those of INa1.
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Methods |
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The Purkinje strands were digested at 37.5°C in the ‘dissociation solution’, which was the Ca2+-free Tyrode solution to which collagenase (1 mg ml–1, type VIII, Sigma) and essentially fat-free bovine serum albumin (2 mg ml–1) had been added. An aliquot of 50 µl of 2.5 mM CaCl2 was added to the 5 ml digestion solution. During the digestion, the strips were shaken in a thermostated bath. The fibres were digested for a total of 40 min, and the strands, cut into 2–3 mm segments, were shaken in the same dissociation solution using a mechanical ‘triturator’ that passed the fibres repeatedly through a pipette tip at 37.5°C. The rate and patterns of agitation were controlled by a Z-80-based microprocessor interfaced to a linear actuator and piston/cylinder (Datyner et al. 1985).
The solution was sampled for the presence of single Purkinje cells under microscopic examination, and trituration repeated as needed. The final suspension was centrifuged at 50g for 5 min, the supernatant discarded, and the pellet resuspended in a Kraft-Brühe (KB) solution of the following composition (mM): KCl, 85; KH2PO4, 30; MgSO4, 5.0; glucose, 20; pyruvic acid, 5.0; creatine, 5.0; taurine, 5.0; EGTA, 0.5; hydroxybutyric acid, 5.0; succinic acid, 5.0; and ATP, 1.1 mg ml–1. The pH was adjusted to 7.2 with KOH.
The single cells thus obtained were stored in KB solution for about 60 min at room temperature. A sample of the cell suspension was placed in a recording chamber located on the stage of an inverted microscope (Nikon Diaphot) equipped with Hoffman modulation contrast optics (x200 magnification). The cells were allowed to settle to the glass bottom of the chamber and then were superfused with Tyrode solution of the following composition (mM): NaCl, 140; KCl, 5.4; MgCl2, 1; CaCl2, 1.8; glucose, 5.5; and Hepes, 5 (pH adjusted to 7.4 with NaOH).
We employed the whole cell patch clamp technique using an Axopatch 1D amplifier (Axon Instruments Inc.). The pipettes were prepared by means of a Narishige PP-83 Glass Microelectrode Puller. The pipettes had a resistance of 2–4 M
when filled with the following solution (mM): potassium aspartate, 100; KCl, 30; MgCl2, 2.0; EGTA, 11.0; Na-Hepes, 10.0; Na2-ATP, 2.0; NaGTP, 0.1; and CaCl2, 5.0. The pH was adjusted to 7.2 with KOH.
The pipette resistance usually increased two- to threefold in the whole cell configuration. The liquid junction potential between the pipette solution and the Tyrode solution was 9 mV (pipette side negative). Since the exchange is never complete because of membrane transport, no correction was made for this effect. On the assumption of 5–8 M series resistance and a maximal current of 1 nA in 5.4 mM extracellular [K+] for INa2, the offset owing to series resistance was at most 8 mV. This offset is in the opposite direction to the liquid junction offset and again no correction was applied. The single Purkinje cells were studied under control conditions in the absence of any channel blocker (Ba2+, Cs+, Mn2+, Cd2+, TTX, etc.). Some of these blockers were tested only after the control recording had been carried out. Successive command steps of the same protocol were applied at intervals of at least 5 s.
Data were analysed by means the pCLAMP program (Axon Instruments Inc.). Depolarizing steps from different holding potentials (Vh) were applied to activate voltage- and time-dependent currents, and ramps were applied, usually between –90 and +40 mV, to study the current–voltage (I–V) relation. The amplitude of the slowly decaying component of INa2 was measured as the difference between the point where the extrapolated decay of INa1 and the backward extrapolation of INa2 met and the current at the end of the step. The region of negative slope during the ramps was measured as the difference between its beginning and its end. With depolarizing steps (but not with ramps), the amplitude of INa1 was often in excess of 10 000 pA, beyond which value the amplifier was saturated. In the figures, during depolarizing steps, INa1 is only partly shown owing to its large size.
The voltages and currents recorded were stored in the computer hard disk and were analysed and fitted by using the Clampfit program (version 6.05, Axon Instruments Inc.). The slowly inactivating current traces were fitted using the Chebyshev technique within pCLAMP software with bi-exponential function according to eqn (1):
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| (1) |
1 and 2 are the time constants, and C is the offset constant. The data were plotted by either Microsoft Excel or Sigmaplot 3.0 and are presented as means ±
S.E.M. Student's t test (Microsoft Excel or Sigmaplot) between two terms of comparison and one-way ANOVA between a data group were applied, and P < 0.05 was considered significant (marked in the text by an asterisk, *); n.s. indicates a statistically not significant difference. |
Results |
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To explore whether the threshold voltage of INa2 is different from that of INa1, steps of 500 ms duration were applied from a Vh of –90 mV to +30 mV in increments of 10 mV (see protocol in Fig. 1).
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With Vh –90 mV, during depolarizing steps to –50 mV, INa1 rapidly activated and inactivated, and was not followed by a time-dependent current. At –40 mV, quite often there was a small inward component which decayed before the end of the step. At –30 mV or less negative values, INa2 decayed throughout the 500 ms step, and the inactivation was usually incomplete by the end of the step. In n = 9 cells, the maximal amplitude of the slowly decaying INa2 was 956.4 ± 111.6 pA at an average potential of –25.5 ± 1.7 mV.
Thus, INa1 activates and inactivates quickly at a potential (–50 mV), which is negative to the threshold for INa2. Only at less negative voltages did the slowly decaying INa2 appear.
Dependence of INa2 on the holding potential
If INa2 is a sodium current, then its amplitude might decrease at less negative holding potentials (Vh), which would decrease a sodium (but not a Ca2+) current. Thus, in single myocytes, a Vh of –50 mV abolishes INa1, but it has no effect on slow inward current (ICa) (Isenberg & Klöckner, 1982).
In Fig. 2, Vh was set at –90 (Fig. 2A), –80 (Fig. 2B), –70 (Fig. 2C), –60 (Fig. 2D), –50 (Fig. 2E) and –40 mV (Fig. 2F) for 3–5 min, and successive depolarizing steps were applied in increments of 10 mV (see protocol in Fig. 2A). As indicated by the arrows, INa2 gradually decreased as Vh was decreased from –90 to –80 and to –70 mV. At Vh –60 and –50 mV, INa2 was no longer present during the depolarizing steps. When, Vh was decreased to –40 mV, a different inward component appeared which activated more slowly and inactivated more quickly than INa2. The differences between the two currents are emphasized in the boxed inset b by the grey areas.
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In the boxed inset a, the graph shows that the maximal amplitude of INa2 during the test potentials (Vtest) decreased with less negative Vh (*, ANOVA). In n = 9 cells, with respect to the maximal value on depolarization from Vh –90 mV, with Vh –80 mV the maximal amplitude of INa2 decreased by –10.6% (at –28.8 ± 1.1 mV) and with Vh –70 mV by –45.5% (at –27.7 ± 1.5 mV). With Vh –60 mV, INa2 was present in only two experiments and it was smaller by –67.7% (at –20.0 ± 0.0 mV). Thus, the maximal INa2 measured at approximately the same Vtest gradually decreased with less negative Vh. These values of Vh would have little effect on ICa, since ICa decreases only with Vh less negative than –50 mV (by 5–10% with Vh –40 mV; Isenberg & Klöckner, 1982). As for INa1, it was still present with Vh –60 mV, although the threshold was shifted to –40 mV and the current was no longer truncated in half of the experiments. With Vh –50 mV, INa1 was present only in two experiments and it was much smaller.
Time constants of the decay of INa2
The INa2 trace during the 500 ms steps was fitted with a bi-exponential function to obtain an estimate of the time constants of INa2 inactivation.
In Fig. 3A, as usually found, there was no time-dependent component during the depolarizing step at –60 mV and INa1 activated and inactivated quickly and completely at –50 mV. Once INa1 had inactivated, the subsequent steady current during the step overlapped that recorded during the pulse at –60 mV (which did not initiate INa1). Instead, during the step at –30 mV, the slowly decaying INa2 appeared, which was still decaying by the end of the 500 ms step (see shaded area). In Fig. 3B, INa2 decay was fitted with a double exponential function: 1 was 7.1 ms and 2 was 220.6 ms.
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1 were not significant, 2 became significantly larger at less negative values of Vtest (*, ANOVA). With different Vh, the time constants during the Vtest at –20 mV were: with Vh
–90 mV, 1
= 8.6 ± 0.6 ms and 2
= 272.3 ms; with Vh
–80 mV, 1
= 8.5 ± 1.3 ms and 2
= 246.0 ± 50.8 ms; and with Vh
–70 mV, 1
= 9.8 ± 1.7 and 2
= 224.4 ± 24.3 (n.s.) In n
= 7 cells with Vh
–80 mV, the time constant (1) of INa1 inactivation during the step at –50 mV was 4.5 ± 1.0 ms. Thus, the slow time constant of inactivation in the voltage range where INa2 is maximal is of the order of 250 ms As expected, the time constants were not significantly affected by the different values of Vh. Because of the voltage range in which it appears and of its slow inactivation, INa2 appears suitable to play a role in the maintenance of the long plateau of Purkinje cells, which usually occurs between 0 and –30 mV (Draper & Weidmann, 1951). Negative slope and voltage-dependent inactivation of INa2
Since INa2 inactivates slowly, it might be expected to cause a region of negative slope in the I–V relation. If so, the negative slope should decrease with lower Vh, since the results reported above indicate that INa2 channels undergo voltage-dependent inactivation. The current–voltage relation was studied by applying depolarizing ramps from different values of Vh. Repolarizing ramps were also applied, since an incomplete inactivation of INa2 at the peak of the ramp may cause a region of positive slope (but not INa1) during the repolarizing ramp. Another advantage of the ramps is that (owing to its partial voltage- and time-dependent inactivation), INa1 may be small enough not to be truncated by the saturation of the amplifier. It should be noted that the amplitude of the ramps was the same at different values of Vh (so that their slope was absolutely the same), but, with different values of Vh, INa2 appeared in the usual potential range. Therefore, INa2 amplitude with different values of Vh could be compared.
In Fig. 4A, with Vh of –90 mV, during the depolarizing ramp (protocol shown in Fig. 4E) there was a degree of inward rectification of the outward current, and at –46 mV INa1 quickly activated (horizontal arrow) and inactivated. The inactivation of INa1 was followed by a pronounced region of negative slope (between dot and oblique arrow). The outward current then re-increased toward its peak. During the repolarizing ramp, there was no INa1, but a region of positive slope was present. The positive slope was smaller and less steep than the negative slope during the depolarizing ramp, but it was within the voltage range of the latter and it was not preceded by INa1. The presence of a positive slope is consistent with an incomplete inactivation of INa2 at the peak of the ramps, so that on repolarization INa2 temporarily diminishes the declining outward current. As INa2 became smaller as a function of voltage, the I–V relation became briefly more outward (positive slope).
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) and more so (
) when Vh was decreased to –70 mV (Fig. 4C). With the less negative Vh, INa1 also decreased to a smaller extent (horizontal arrows), and at Vh
–60 mV it was rudimentary with no negative slope (Fig. 4D). The traces labelled with •, and were superimposed in the boxed inset by the beginning of the negative slope and emphasize the decrease in amplitude of the negative slope with the less negative Vh. During the repolarizing ramp, the amplitude of the positive slope also decreased with the less negative holding potentials. At Vh –60 mV, the positive slope was no longer present. These findings suggest that the negative slope on depolarization and the positive slope on repolarization result from a slowly inactivating component carried by Na+ (but not by Ca2+).
As shown by graph in Fig. 4 (n = 11 cells), the amplitude of the negative slope was 165.5 ± 53.5, 136.0 ± 40.6, 88.6 ± 30.0 and 17.7 ± 7.2 pA with Vh –90, –80, –70 and –60 mV, respectively. Thus, with respect to the value with Vh –90 mV, the negative slope amplitude decreased by –17.8*, –46.4* and –89.3%* with Vh –80, –70 and –60 mV, respectively. When calculated from the percentage change of the single tests with respect to the value with Vh –90 mV, the decrease of the negative slope amplitude was significant (*, ANOVA).
The findings indicate that INa2 is smaller during a ramp than during a depolarizing step and also that it decreases with less negative holding potentials. As for INa2 versus INa1, the amplitude of the negative slope was 6.7, 5.0 and 3.1% of INa1 with Vh –90, –80 and –70 mV, respectively. At –60 mV, INa2 was 30.2% of INa1 (n = 6), the larger percentage being due to the marked decrease of INa1. In these tests, INa1 was smaller than the value at which it is cut off by the saturation of the amplifier. Therefore, the slowly inactivating INa2 (the fraction of INa2 that is important for the plateau duration) is a small percentage of INa1 (less than 10%) and becomes an even smaller at less negative Vh.
The effects of tetrodotoxin and lignocaine on INa2
If INa2 is a sodium (and not a calcium) current, then the sodium channel blockers TTX and lignocaine may reduce or suppress INa2.
In Fig. 5A and B, INa1 appeared during the depolarizing step at –50 mV and INa2 (arrow and ) reached its maximal value at –20 mV. In Fig. 5C and D, when the same procedures were repeated in the presence of TTX, INa1 decreased from the control value of 9578 to 2842 nA (–70.3%) and INa2 was suppressed (•). The difference current (control minus TTX) is shown in Fig. 5E.
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In Fig. 6, INa1 appeared at –50 mV whereas INa2 was maximal at –30 mV (arrow in Fig. 6A and 
in Fig. 6B). In Fig. 6C and D, lignocaine (100 µM) markedly reduced INa2 () when INa1 was still big enough to be truncated by the saturation of the amplifier, as in control conditions. When Vh was reduced to –60 mV, INa2 decreased even in Tyrode solution and yet INa1 was still saturated. When lignocaine was added, INa2 was nearly abolished and INa1 was markedly reduced (not shown).
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Effects of faster ramps on INa2 in the absence and presence of TTX
If due to INa2, then the negative slope may become larger with faster ramps (less inactivation during depolarization) and it should be sensitive to blockade by TTX.
In Fig. 7, 500 (Fig. 7A), 250 (Fig. 7B) and 125 ms depolarizing ramps (Fig. 7C) were applied from Vh –90 mV to +40 mV. During the depolarizing ramps, INa1 was followed by the negative slope whose peak is indicated by the oblique arrows.
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Tetrodotoxin markedly decreased the magnitude of the negative slope (trace marked by in Fig. 7D; see shaded area in the boxed inset b) and more so the faster the ramps (Fig. 7E and F). Tetrodotoxin suppressed the small negative slope during the 10 s depolarizing ramps (not shown).
In n = 4 cells, with the 500 ms depolarizing ramps, the negative slope size was 310 ± 88 pA in control conditions and 56 ± 13 pA* (–81.9%) in the presence of TTX (30 µM). The corresponding values for INa1 were 5771 ± 664 and 970 ± 808 pA* (–83.1%). Thus, INa2 was 5.3% of INa1 in control conditions and 5.7% of INa1 in the presence of TTX.
With the 250 ms ramps, the negative slope size was 496 ± 149 pA in control conditions and 6 ± 2 pA* (–98.7%) in the presence of TTX. The corresponding values for INa1 were 6594 ± 1005 and 1434 ± 679 pA* (–78.2%). Thus, INa2 was 7.5% of INa1 in control conditions and 0.4% of INa1 in the presence of TTX.
With the 125 ms ramps, the negative slope size was 724 ± 263 pA in control conditions and 8 ± 2 pA (–98.8%, P = 0.05) in the presence of TTX. The corresponding values for INa1 were 7502 ± 801 and 2266 ± 1023 pA* (–69.7%). Thus, INa2 was 9.6% of INa1 in control conditions and 1.1% of INa1 in the presence of TTX. These results indicate that the magnitude of the negative slope is time dependent (in contrast to the window current) and that INa2 is more sensitive than INa1 to the inhibitory effect of TTX.
Effects of cadmium administration on INa2
Cadmium blocks INa1 (DiFrancesco et al. 1985) and therefore its effects on INa2 were also tested. In Fig. 8A, during the step to –50 mV, INa1 was quickly activated and inactivated and the remainder of the current trace was superimposed on that recorded during the pulse to –60 mV (no slow component). Instead, at –30 mV there was a large INa2 (•), as usual. In Fig. 8B, in the presence of Cd2+ (0.2 mM), both INa2 and INa1 were suppressed. In the boxed inset, the superimposed traces recorded during the step at –30 mV in control conditions (•) and in the presence of Cd2+ () emphasize the suppression of INa2 and INa1 by Cd2+ (shaded area). In Fig. 8C, a larger depolarization (–20 mV) elicited a small INa1 (shaded area), whereas INa2 was still blocked.
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Effects of manganese on INa2
In addition to INa1, Cd2+ also blocks the slow inward current, whereas manganese blocks ICa but not INa1 (see DiFrancesco et al. 1985). Therefore, in contrast to cadmium, manganese would not be expected to block INa2 if it is a slowly inactivating Na+ current.
In Fig. 9A, in Tyrode solution, a depolarizing step from Vh
–90 mV to –50 mV elicited the rapidly activating and inactivating INa1 (not shown) and that to –30 mV elicited INa2 as well. In Fig. 9B, manganese affected the amplitude and decay of INa2 very little, whereas in the same cell, TTX had its usual inhibitory effects (Fig. 9C). In the boxed inset, the superimposed traces emphasize the very little difference between the control (•) and manganese traces (), and the abolition of INa2 by TTX ( and shaded area).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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These results indicate that INa2 is unlikely to be a fraction of INa1 channels that inactivate slowly, since, at its threshold voltage, INa1 was not followed by INa2, the latter appearing only at less negative potentials. Several findings indicate that INa2 involves a slowly inactivating Na+ channel, since it was decreased or abolished by a decrease in Vh, by TTX, by lignocaine and by Cd2+, but not by the slow channel blocker Mn2+. The region of negative slope during depolarizing ramps is consistent with the slow inactivation of INa2, since it appeared after the inactivation of INa1 in the voltage range of INa2, increased in size with faster ramps, decreased with a less negative Vh and was blocked by TTX. Many of these characteristics also rule out the possibility that INa2 might be related to the background Na+ or to the window currents, which are not time dependent.
Owing to its characteristics, the slowly inactivating component of INa2 would contribute to the long plateau of Purkinje fibres, since the voltage range of the plateau (from about 0 to about –30 mV; Draper & Weidmann, 1951) is similar to that where INa2 appears. The greater sensitivity of INa2 to TTX relative to INa1 may account for the fact that in Purkinje fibres TTX at suitable concentrations shortens the plateau (Coraboeuf et al. 1979; Bhattacharyya & Vassalle, 1982) more than it decreases the amplitude and the rate of rise of the upstroke (Coraboeuf et al. 1979). The positive slope during the repolarizing ramp is of interest also in regard to a possible role of the inactivating INa2 in the induction of early after-depolarizations.
The relation between INa2 and INa1
The conclusion that INa1 and INa2 are related to two different sodium channel isoforms with different thresholds and different inactivation kinetics is based on the fact that the two currents can appear separately. The difference in thresholds (–50 mV for INa1 and –40 mV or less for INa2) is not consistent with INa2 being due to the slow inactivation of a fraction of the fast INa1 channels. This is supported by the fact that, with sufficiently slow ramps, a small INa2 can be present in the absence of INa1 and that during the repolarizing ramps a slow inward component (but not INa1) is present.
The overlapping of the traces during the steps at –60 and –50 mV (after the fast inactivation of INa1) suggest that there was no loss of voltage control to influence INa2 recorded at less negative potentials. This is supported by the immediate fall of the slope conductance after the fast inactivation of INa1 at –50 mV and by the large and decaying slope conductance during INa2 (Bocchi & Vassalle, 2000).
The findings suggest that INa2 activates quickly, since INa1 was not prolonged by INa2; only during the late inactivation of INa1 did the slow decay of INa2 appear. Therefore (when its threshold is attained) INa2 should rapidly activate along with INa1, as indeed shown by the fact that the two currents can be separated by applying a two step protocol: during the first step to –50 mV, INa1 activated and inactivated (no INa2) and during the second step from –50 mV to less negative values, a smaller INa2 quickly activated and then slowly inactivated (Bocchi & Vassalle, 2000).
The ionic species carrying INa2
The various tests carried out show that INa2 behaves like a sodium current with no apparent contribution of the calcium current, ICa. Thus, during depolarizing steps and the negative slope, INa2 was reduced by less negative holding potentials (–60 to –50 mV) that have little effect on ICa (Isenberg & Klöckner, 1982). Indeed, with a Vh of –60 mV, INa2 may no longer be present and yet with a Vh of –40 mV, an inward current appeared on depolarization which decayed in about 100 ms, as ICa does (Isenberg & Klöckner, 1982). Furthermore, the slower time constant of inactivation of INa2 (2
=
–250 ms at –30 to –20 mV) is much greater than that of ICa (2
= 30 ms for a step to 0 mV; Isenberg & Klöckner, 1982).This conclusion is substantially supported by the reduction or suppression of INa2 by TTX, lignocaine and cadmium. The absence of a Ca2+ contribution to INa2 is also shown by the failure of manganese to decrease INa2, in agreement with the finding that TTX shortens the Purkinje cell action potential to a similar extent in the absence and presence of verapamil (Coraboeuf et al. 1979). Also, the shortening of action potential duration by TTX (Bhattacharyya & Vassalle, 1982) and INa2 (Bocchi & Vassalle, 2000) are little affected by changes in extracellular [Ca2+]. In addition, a TTX concentration that blocks INa1 does not alter ICa (Isenberg & Klöckner, 1982).
Slow sodium currents in cardiac tissues
Slowly inactivating sodium currents have been reported by several investigators and therefore the question arises of whether INa2 is one of the currents that has been already described.
In this connection, a distinction should made between Purkinje fibres and ventricular muscle fibres. Thus, the action potential of Purkinje fibres is much longer than that of ventricular muscle fibres (see Vassalle & Bhattacharyya, 1980; Bhattacharyya & Vassalle, 1982). This difference seems to be related to INa2 (rather than to some other currents) for the following reasons: (i) TTX shortens the action potential of Purkinje fibres much more than that of ventricular muscle fibres (Coraboeuf et al. 1979; Bhattacharyya & Vassalle, 1982; Iacono & Vassalle, 1990); (ii) TTX decreases intracellular sodium activity much more in Purknje fibres than in myocardial fibres (Iacono & Vassalle, 1990); (iii) local anaesthetics shorten the action potential of Purkinje fibres but not that of ventricular muscle (Vassalle & Bhattacharyya, 1980); and (iv) there is very little or no INa2 in ventricular myocardial fibres (Bocchi & Vassalle, 2000).
These findings imply that results obtained in ventricular myocardial cells cannot be extrapolated ipso facto to Purkinje fibres (and vice versa) and therefore they require a critical appraisal to determine whether currents described in myocardial cells are related to those in Purkinje fibres. Furthermore, possible species differences need to be considered; for example, the action potential of Purkinje fibres is strikingly different from that in rat ventricular myocardial fibres (especially in the plateau range of potentials). In addition, different methodological approaches may influence the results obtained. Finally, the currents described need to be consistent with the electrophysiological characteristics of the action potentials if they in fact play a role in determining their shape and duration.
For example, in rat ventricular muscle, a persistent sodium current inactivates only slightly during a depolarizing step lasting up to 900 ms and it is little affected by a prepulse at –50 mV (Saint et al. 1992), whereas INa2 starts inactivating when INa1 inactivation is nearly completed and it is decreased by a less negative Vh. The myocardial persistent current begins activating at potentials negative to –90 mV from a Vh of –130 mV, whereas INa2 activates at –40 mV or less negative values. Also, there is no plateau in rat ventricular muscle in the range where it is present in Purkinje fibres.
In another study (Richmond et al. 1998), the slow inactivation of Na+ channels in normal and mutant human cardiac muscle was investigated in channels expressed in oocytes. Several considerations preclude a similarity between the slow inactivation of those Na+ channels and that of INa2. For example, ventricular tissues were used, and slow inactivation required depolarization to positive potentials for 60 s in normal or in mutant channels. The pulses used in the protocols lasted tens of seconds and involved potentials as negative as –150 mV. Zilberter et al. (1994) studied single channels in both rabbit Purkinje fibres and ventricular muscle. On depolarization from –120 to –40 mV, the late openings of sodium channels lasted for several seconds (up to 5 s). Also, these channels were qualitatively similar in both cell types, whereas INa2 is generally absent in myocardial cells (Bocchi & Vassalle, 2000). The late openings of these Na+ channels were studied at –40 mV, a potential at which INa2 is very small and may not be present in every Purkinje cell. Also, the late current appears to involve the background Na+ channels (Zilberter et al. 1994).
Studies conducted in Purkinje fibre strands (canine, Gintant et al. 1984; rabbit, Carmeliet, 1987) are much more pertinent to the present report. Indeed, the I–V relation measured during depolarizing ramps showed a slowly inactivating TTX-sensitive current, beginning at negative voltages (aproximately –80 mV, Gintant et al. 1984; –90 mV, Carmeliet, 1987) and extending to positive values. Gintant et al. (1984) do point out that this current would be important for the duration of the plateau, and Carmeliet (1987) indicates that it would be important for both diastolic depolarization and plateau duration. However, INa2 begins to activate at –40 mV, peaks at –30 to –20 mV and is much smaller than INa1. One possible reason for the differences (apart from the different voltage clamp technique used by us; single cells versus non-dissociated fibres) might be the presence of another slowly inactivating sodium current (INa3, Rota & Vassalle, 2003) with a threshold of about –60 mV and which may be important for the late diastolic depolarization (see Rota & Vassalle, 2003).
Possible relation of INa2 to the molecular biology of slowly inactivating sodium channels
Since INa2 has characteristics that are different from those of INa1, the question needs to be addressed of whether INa2 might be related to known sodium channel isoforms that exhibit slow inactivation.
So far, nine different Na+ channels (NaV1.1 to NaV1.9) have been identified and functionally expressed (Catterall et al. 2005). The three kinds of sodium channels (cardiac, neuronal and skeletal) present in cardiac tissues have different characteristics (and presumably different functions). One major difference is that in the heart, the neuronal Na+ channel isoforms (NaV1.1, NaV1.2, NaV1.3 and NaV1.7) as well as the skeletal muscle isoform (NaV1.4; Catterall et al. 2005) are more sensitive to tetrodotoxin than the cardiac isoforms (NaV1.5, NaV1.8 and NaV1.9). Of the cardiac isoforms, NaV1.5 is believed to generate the bulk of INa1, with other channels contributing a smaller fraction of Na+ entry (Haufe et al. 2005a,b).
In addition to different TTX sensitivity and different contribution to Na+ entry, different sodium channels display fast or slow inactivation. The inactivation of INa1 (mostly due to NaV1.5) is fast, whereas that of other channels (including the skeletal muscle NaV1.4) is slow (Vilin et al. 1999). This raises the question of whether NaV1.4 might be among the possible Na+ channels that underlie INa2.
An indispensable prerequisite for such a possibility is that NaV1.4 should be expressed also in Purkinje fibres and not only in myocardial tissues (Vilin et al. 2001; Zimmer et al. 2002; Haufe et al. 2005a and b,,). In fact, expression and localization of NaV1.4 protein in canine Purkinje fibres has recently been demonstrated by confocal microscopy on isolated cells; a clear staining, most prominent on cell sarcolemma, was observed for NaV1.4. In contrast, NaV1.5 shows remarkable striation staining pattern. Also, other preliminary data are consistent with the expression of NaV1.4 mRNA in canine Purkinje cells (Y. Qu, M. Chahine, M. Vassalle & M. Boutjdir, unpublished observations).
It is suggestive that NaV1.4 inactivation is slowed by veratridine (Wang & Wang, 1998) and by sea anemone toxin ATX II (Chahine et al. 1996). Similarly, the action potential of Purkinje fibres is prolonged by both veratridine (Iacono & Vassalle, 1990) and ATX II (El-Sherif & Turitto, 2003). The NaV1.4 channel is blocked by TTX, local anaesthetics and lignocaine (Makielski et al. 1999), which also shortens and shifts to more negative values the plateau of Purkinje fibres (Vassalle & Bhattacharyya, 1980; Bhattacharyya & Vassalle, 1981, 1982). However, experimental proof for a role of NaV1.4 in INa2 is not available.
Conclusions
The function of the longer action potential in Purkinje fibres appears to be the prevention of the re-entry of excitation from the activated ventricular myocardium (Myerburg et al. 1970). The present results show that on depolarization and with a threshold positive to that of INa1, a current (INa2) flows that would indeed prolong the plateau and that has the characteristics of a sodium current.
The results suggest that INa2 is unlikely to be related to a cardiac NaV channel, since cardiac NaV channels show a rapid activation and inactivation, as seen for INa1 at its threshold. As for the TTX-sensitive slowly inactivating NaV channels, lack of evidence prevents the drawing of conclusions on the involvement of neuronal versus skeletal muscle NaV channels. The data presented do suggest that INa2 is due to a Na+ isoform that is different from that of INa1 (NaV1.5). The identification of the slowly inactivating isoform responsible for INa2 will require further experimentation
On the basis of the collected evidence, it may not be too hazardous to hypothetize that the nine sodium isoforms already identified and expressed in cardiac tissues may be involved in different functions of different cardiac cells, such as pacemaking (INa3), duration of the action potential (INa2), excitation and conduction (INa1) and, indirectly, contraction.
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