The Na+ channel is voltage gated and characterized by three distinct states: closed, open, and inactivated. To identify the effects of halothane on the cardiac Na+ current (I(Na)) at various membrane potentials, the effects of 1.2 mM halothane at different holding potentials (V(H)) on I(Na) were examined in single, enzymatically isolated guinea pig ventricular myocytes.
The I(Na) was recorded using the whole-cell configuration of the patch-clamp technique. Currents were generated from resting V(H)s of -110, -80, or -65 mV. State-dependent block was characterized by monitoring frequency dependence, tonic block, and removal of inactivation by veratridine.
Halothane produced significant (P < 0.05) V(H)-dependent depressions of peak I(Na) (mean +/- SEM): 24.4 +/- 4.1% (V(H) = -110 mV), 42.1 +/- 3.4% (V(H) = -80 mV), and 75.2 +/- 1.5% (V(H) = -65 mV). Recovery from inactivation was significantly increased when cells were held at -80 mV (control, tau = 6.0 +/- 0.3 ms; halothane, tau = 7.1 +/- 0.4 ms), but not at -110 mV. When using a V(H) of -80 mV, halothane exhibited a use-dependent block, with block of I(Na) increasing from 8.6 +/- 1.4% to 30.7 +/- 3.5% at test pulse rates of 2 and 11 Hz, respectively. Use-dependent inhibition was not apparent at V(H) of -110 mV. When inactivation of I(Na) was removed by exposure to 100 microM veratridine, no significant difference was observed in the depressant effect of halothane at both V(H)s: 26.6 +/- 4.5% (V(H) = -80 mV) and 26.4 +/- 5.6% (V(H) = -110 mV).
The present findings indicate that the depressant action of halothane on cardiac I(Na) depends on the conformational state of the channel. As more channels are in the inactivated state, the more potent is the effect of halothane. Removal of channel inactivation by veratridine abolished the dependence of the halothane effect on V(H), but depression of the current was still evident. These results indicate a complex interaction between halothane and the various conformational states of the Na+ channel.
Halothane, at clinically relevant concentrations, depresses cardiac conduction consistent with reductions in maximum voltage (Vmax) of phase 0, action potential amplitude, and overshoot. [1–3] This depression of cardiac conduction may be responsible for inducting reentrant dysrhythmias that are associated with halothane administration, [1,4] and they may be attributed to a depression of the fast inward Na sup + current (INa) during the action potential upstroke of the ventricular muscle fiber.  In a recent study, halothane, at clinical concentrations, depressed cardiac Na sup + current in a concentration- and voltage-dependent manner.  However, the underlying mechanisms of interaction between halothane and the cardiac Na sup + channel are unclear. 
The cardiac Na sup + channel is voltage gated and characterized by three distinct states: closed, open, and inactivated. Under physiologic conditions, where the ventricular muscle diastolic membrane potential is approximately -80 to -90 mV, a fraction of the available Na sup + channels is inactivated. During the acute phase of myocardial ischemia, ventricular cells may be depolarized to between -50 and -60 mV due to accumulation of extracellular potassium. Such an effect would further increase the fraction of Na sup + channels in the inactivated state. [7,8] Observations that halothane reduced overshoot and Vmaxin the infarcted but not in the normal Purkinje fibers  suggested that there were fewer channels available for activation.  It was also hypothesized that halothane may have a greater affinity for inactivated Na sup + channels than for those in the active or resting state.  This hypothesis was further supported by results showing that the depressant effect of halothane on INawas more pronounced at depolarized membrane potentials. Halothane also induced a hyperpolarizing shift in steady-state inactivation. 
The present study was designed to better characterize these voltage-dependent effects of halothane on cardiac Na sup + current and to identify a possible mechanism of interaction. Using the whole-cell patch-clamp method, we tried to determine whether halothane block depends on the various channel conformations, particularly the inactivated state.
After we received approval of the institutional animal care and use committee, single ventricular myocytes were enzymatically isolated from adult guinea pigs that weighed 200–300 g. The procedure for cell isolation is the same as that described previously. [6,13] Briefly, single cardiac myocytes were obtained by retrograde perfusion of guinea pig hearts with an enzyme (collagenase; Gibco, Life Technologies, Grand Island, NY). Isolated cells were transferred to a plexiglass chamber mounted on the stage of an inverted microscope (model IMT-2; Olympus, Tokyo, Japan). Only rod-shaped cells with clear borders and striations were selected for experiments, and they were used within 12 h of isolation.
High-resistance seals and voltage clamp were attained in Tyrode solution containing 132 mM NaCl, 4.8 mM KCl, 1.2 mM MgCl2, 10 mM HEPES, 5 mM dextrose, and 1 mM CaCl2while pH was adjusted to 7.4 with the addition of NaOH. After whole-cell voltage clamp was established, the external bath solution was changed to one that isolated the Na sup + channel current from other membrane currents. For experiments initiated from a VHof -110 mV and -80 mV, the solution contained 10 mM Na sup +, 115 mM CsCl, 10 mM HEPES, 1 mM MgCl2, 1.8 mM CaCl2, 5.5 mM glucose, and 3 mM CoCl; pH, 7.2 with CsOH. Cobalt and cesium were used to block L-type calcium and potassium channels, respectively. In experiments starting from a VHof -65 mV, the external Na sup + concentration was increased to 100 mM to achieve a comparably sized Na sup + current (between 1.5 and 2.3 nA). To compensate for changes in the osmolarity of the solution, CsCl was decreased to 25 mM. The standard pipette solution contained 11 mM EGTA, 1 mM CaCl2, 10 mM HEPES, 2 mM Mg-ATP, 90 mM CsF, 60 mM CsCl, 10 mM NaF; pH, 7.3 with CsOH. Repetitive current-voltage measurements were obtained while myocytes were exposed to control and test solutions to monitor time-dependent changes in INa. The final bath concentration of halothane used was 1.2 mM, which is equivalent to 1.96 vol% in the gas phase at 22 [degree sign] Celsius in Tyrode solution.  The anesthetic solutions were applied via glass syringes (50 ml) using a peristaltic pump (flow rate, 1.5 ml/min). Samples of the solution in the chamber were taken after every experiment and analyzed by gas chromatography to verify the anesthetic concentration surrounding the cell.
Recording Procedures and Data Analysis
Current measurements were obtained in the whole-cell configuration of the patch-clamp procedure as described by Hamill et al.  To optimize voltage control of the cardiac cells, (1) small cells were selected to ensure that membrane capacitance would be small (60–80 pF);(2) the external Na sup + concentration was reduced (10 or 100 mM, depending on VH) to decrease the magnitude of Na sup + current (between 1.5 and 2.3 nA);(3) pipette resistances ranged from 1.0 to 1.5 M Omega;(4) experiments were performed at room temperature (22 [degree sign] Celsius); and (5) series resistance compensation was adjusted (about 80%) to give the fastest possible capacitance transients without causing ringing. Under these conditions, the voltage error was < 3 mV. Pipettes from borosilicate glass were pulled using a multiple-stage puller (model PC-84; Sachs-Flaming, Sutter Instruments, Novato, CA) and heat polished (Narishige microforge, model MF-83, Tokyo, Japan). Current was measured using a List EPC-7 patch-clamp amplifier (Adams & List Associates, Great Neck, NY). Current was digitized at 67 kHz and lowpass filtered at 3 kHz. All data were digitized and stored for later analysis using the pCLAMP package (Axon Instruments, Foster City, CA).
After rupturing the membrane and achieving voltage clamp conditions, the cell was allowed to equilibrate for at least 15 min before experimental measurements were initiated. No visible change of the current amplitude for 4 min was used as a criteria for stable current before recordings were initiated. To determine the peak INa, whole-cell currents were elicited with 30-ms test pulses from a VHof -110, -80, or -65 mV to +30 mV in 10-mV increments. To monitor the time-dependent changes in peak INa, 50-ms test pulses were applied every 15 s to -20 mV when peak INaoccurred.
To monitor steady-state inactivation of INa, cells were subjected to 500-ms preconditioning pulse potentials between -130 (the V sub H) and +20 mV and then stepped to the test pulse potential of -20 mV (24 ms), where peak Na sup + current occurred. Peak currents evoked by the test potentials were normalized to the maximum current (Imax) obtained during the test potentials. Steady-state inactivation was fitted to a Boltzmann distribution  described by:Equation 1where V is the preconditioning potential, V1/2 is the potential at which half-maximum inactivation occurred, and k is the slope factor. The double-pulse protocol was used to determine recovery from inactivation. A 100-ms reference pulse to -20 mV was followed by a variable recovery interval at -110 or -80 mV before a second test pulse to -20 mV (10 ms duration) was applied.
Results are expressed as means +/- SEM. Statistical differences were determined using the paired Student's t tests when a cell served as its own control. One-way analysis of variance was used when different groups of data were compared. Differences between group means were evaluated using the Bonferroni test. Statistical analysis was computed using commercially available software (SigmaStat; Jandel Scientific, Corte Madera, CA). Test results were considered significant when P < 0.05.
Depression of Cardiac Na sup + Current by Halothane Depends on the Fraction of Available Channels in the Inactivated State
Depression of the peak Na sup + current by halothane (1.2 mM) depended on the VH(Figure 1). At the more depolarized VH, the effect of halothane was greater. At a VHof -65 mV, where most of the channels are in the inactivated state, and, therefore, only a few channels are available for activation, the suppression of INaby halothane was most pronounced with an average reduction of 75.2 +/- 1.5%. The effect of halothane at the three tested VHs occurred within seconds of exposure to halothane and reached a steady state after approximately 3 min. A complete reversal of the halothane effects on washout was observed only during experiments conducted from a VHof -110 mV. Recovery of INaat VHs of -80 and -65 mV was approximately 50% and 20%, respectively (data not shown).
Availability of Na sup + channels at three different VHs was determined from the steady-state inactivation of the current. Figure 2shows the steady-state inactivation curve obtained from 12 cells under control conditions. At the hyperpolarized VH(-110 mV), all of the Na sup + channels were available for activation. At the more depolarized V sub H s, the fraction of channels in the inactivated state increased to 14.00 +/- 0.02% at -80 mV and 78.20 +/- 0.04% at -65 mV.
Reversal of the Halothane Effect Depends on Channel Conformation
(Figure 3) illustrates the time course of the peak INaobtained during control to -20 mV from a VH=-110 mV, followed by another control from VH=-80 mV, halothane (1.2 mM) exposure from V sub H =-80 mV, and the washout. The baseline value between the two controls at VHs -110 mV and -80 mV decreased because of an increased fraction of the available channels in the inactivated state. When the washout was performed at a VHof -80 mV, recovery of the peak INaamplitude was incomplete. Even when the washout period was extended to 20 min (data not shown), full recovery was not achieved. However, when the VHwas set to -110 mV in the same cell, peak INarecovered to control levels (Figure 3(A)).
As shown in Figure 3(B), we next applied a hyperpolarizing conditioning pulse to -110 mV for 3 min (to remove inactivation and allow the channel to recover) before returning to a VHof -80 mV. Again, from -110 mV, recovery from the halothane's effect was rapid and complete. When switching back to a VHof -80 mV during the washout period, recovery was still not complete but more so than without the conditioning pulse from -110 mV.
The length of the -110 mV conditioning pulse (Figure 3(C)) affected the Na sup + current amplitude during the washout at -80 mV. When the period of the conditioning pulse to -100 mV was increased to 7 min, recovery of the INaamplitude at -80 mV was nearly complete (90.8 +/- 6.0%). Thus, as summarized in Figure 3(D), the effect of halothane from a VHof -110 mV was reversible. On the other hand, when using a VHof -80 mV, recovery was significant only after a preconditioning pulse to -110 mV for a prolonged period.
Effects of Halothane on Inactivation Kinetics and Recovery from Inactivation
The results from the holding potential experiments suggest a state-dependent interaction between halothane and cardiac Na sup + channel. Therefore, we investigated whether the channel recovery from an inactivation is affected by halothane. Recovery from inactivation depended on the availability of the Na sup + channels, as determined by the VH, and could be fitted with a single exponential function. From a VHof -80 mV, the rate of recovery of channel inactivation was significantly slower during halothane exposure (77 +/- 5 ms) compared with control (63.2 +/- 4.1 ms;Figure 4(A)). In the presence of halothane and using a hyperpolarizing VH(-110 mV), the rate of recovery from inactivation is only slightly affected without a significant difference from control (Figure 4(B)). The mean recovery time constant was 6.0 +/- 0.3 ms for control and 7.1 +/- 0.4 ms for halothane. The recovery rates for both control and halothane were significantly slower at a VHof -80 mV than -110 mV. Data for recovery rates were obtained in 8–10 cells for each experimental set.
We studied the kinetics of peak INainactivation by fitting a biexponential function to the decay phase of INaduring a 50-ms test pulse to -20 mV. The rate of current inactivation was not significantly affected by halothane at VHs of -110 and -80 mV (n = 16 cells per experimental set). From a VHof -110 mV, the time constants in the control for fast (tauf) and slow (taus) components of INainactivation were 1.45 +/- 0.07 ms and 4.59 +/- 0.38 ms and after halothane exposure they were 1.32 +/- 0.06 ms and 4.72 +/- 0.60 ms, respectively. For experiments starting from a VHof -80 mV, data obtained for taufand tauswere 1.29 +/- 0.04 ms and 4.22 +/- 0.17 ms for control, and they were 1.33 +/- 0.03 ms and 4.01 +/- 0.17 ms during halothane, respectively. The rates of current inactivation (taufand taus) for both control and halothane were significantly different between the VHs of -80 mV and -110 mV, respectively.
Tonic and Phasic Block by Halothane
Two types of block, tonic and phasic, were observed in the inhibition of cardiac Na sup + current by halothane. Tonic block is defined as the decrease in current between an initial pulse after drug exposure compared with an equivalent pulse before drug exposure. We determined the ability of halothane to induce a tonic block by applying 30-ms test pulses to -20 mV from two VHs, -110 mV or -80 mV. Cells were superfused for 4 min with halothane (1.2 mM) while clamped at either -80 or -110 mV. At both VHs, the onset of block during the halothane exposure was significant and complete after the first test pulse (Figure 5), indicating that no channel opening was necessary for the block to occur. The average depression of Na sup + currents from at least six cells was 22.9 +/- 3.3%(VH=-110 mV) and 43.6 +/- 3.9%(VH=-80 mV).
Phasic or use-dependent block is defined as the decrease in current obtained during repetitive depolarizing pulses applied in the presence of the drug. To study the use-dependent block, fifty 50-ms-long pulses were applied at 11 and 20 Hz from a VHof -110 mV or at 2 and 11 Hz from a VHof -80 mV. Different frequencies were used at different VHs to account for the profound inherent frequency-dependent inhibition of the Na sup + channel. Figure 6shows the INavalues recorded from the representative cells. In the absence of halothane, the repetitive depolarization at 2 Hz (VH=-80 mV) and 11 Hz (VH=-110 mV) produced little decrease in INa. During the control conditions, in the absence of anesthetic, at higher stimulation frequency of 11 Hz (VH=-80 mV) and 20 Hz (VH=-110 mV), a use-dependent reduction of INawas observed (Figure 6). In the presence of halothane, a significant use-dependent block of INawas only obtained during the experiments in which the VHwas -80 mV (Figure 7(A)). To describe the magnitude of the block, the last pulse of the train (pulse number 50) was compared with the first pulse.  To demonstrate the development of the use-dependent block as a function of stimulation frequency, the normalized difference [(control - halothane)/control] was plotted against stimulation frequency (Figure 7(B)). There was a significant increase in the percentage of blocked channels in the presence of halothane as the stimulation rate was increased from 2 to 11 Hz at a VHof -80 mV (8.6 +/- 1.4% to 30.7 +/- 3.5%, respectively), as shown in Figure 7(B). At the test pulse rates of 11 and 20 Hz from a VHof -110 mV, the normalized difference in Na sup + current between the control and halothane was not significant (Figure 7(B)).
To examine the effect of halothane under conditions in which only a fraction of the Na sup + channels would be expected to make a transition into the inactivated state, a train of four short (3.5 ms) test pulses to -20 mV was applied from two different VHs (Figure 8). The results show that from a VHof -110 mV, the depression of INaamplitude by halothane was still evident, even with minimal inactivation (Figure 8(A)). The depressant effect of halothane was further enhanced when the protocol was repeated from a VHof -80 mV (Figure 8(B)). Furthermore, when starting from a VHof -80 mV, the development of the block increased significantly with higher pulse number. When plotted as a normalized difference between the test pulses, the inhibition between pulses 1 and 2 was 16.3 +/- 0.9%; pulses 2 and 3, 23.0 +/- 1%; and pulses 3 and 4, 27.6 +/- 1.7%(Figure 7(C)). At a VHof -110 mV, the degree of block remained unchanged between pulses 2 and 4 (Figure 8(C)).
Effects of Halothane on Veratridine-modified Na sup + Current
Our results suggest that one of the mechanisms involved in the effects of halothane depends on the degree of Na sup + channel inactivation. To further test this hypothesis, we studied the effect of halothane in cells in which Na sup + channel inactivation was chemically removed by exposure to veratridine. Under these conditions, the possible interactions between halothane and the inactivated state of the channel were minimized. The neurotoxin veratridine has been shown to induce a long-lasting open state of Na sup + channels in several tissues, including guinea pig ventricular myocytes. Figure 9(A) shows a typical example of the time course of INaafter exposure to veratridine (100 micro Meter). Approximately 6 min after introduction of veratridine to the superfusate, nearly all inactivation of INawas removed. The average decrease of the peak Na sup + current amplitude after veratridine exposure was 29.6 +/- 4.3%(VH-80 mV) and 27.7 +/- 4.2%(VH-110 mV).
When the channel inactivation was removed and a steady-state amplitude of INawas established, halothane depressed INaat both VHsto a similar degree: 24.4 +/- 3.0%(VH-110 mV, n = 7 cells) and 25.9 +/- 3.2%(VH-80 mV, n = 7 cells). Examples of this effect are illustrated in Figure 9(B and C). After washout of halothane and veratridine when the channel inactivation returned, halothane produced a similar depression of 19.8 +/- 1.9% from the hyperpolarizing VHof -110 mV (Figure 9(B)). However, from the VHof -80 mV, with washout of halothane and veratridine when inactivation properties had recovered, the depression of INaby halothane (41.1 +/- 5.4%) was significantly enhanced compared with the effect of halothane in the presence of veratridine (Figure 9(C)).
Our study shows that halothane at a clinically relevant concentration suppressed cardiac Na sup + current in ventricular guinea pig myocytes in a state-dependent manner and supports the hypothesis that halothane interacts with the inactivated state of the Na sup + channel.  The potency of halothane block was much greater when the fraction of channels in the inactivated state was increased by depolarization of V sub H from -110 to -80 mV. When using a holding potential (VH) of -80 mV but not -110 mV, there was significant slowing in the rate of recovery from inactivation with halothane, indicating that halothane may affect the channel conformation between the inactivated and the resting states. Halothane may have a higher affinity to inactivated channels than to channels in the resting state. The results may also suggest that halothane stabilizes an inactivation state of the channel.
The results from the present study, however, indicate that the interaction between halothane and the cardiac Na sup + channel is not confined only to the inactivated state of the channel. Halothane exhibited tonic block in the experiments using holding potentials of -80 and -110 mV, suggesting that no channel opening (and hence no channel inactivation) is required for the block to occur. At -110 mV, halothane might bind to Na sup + channel in its resting (closed) conformation. At -80 mV, when 14% of the Na sup + channels were in the inactivated state, the resting and inactivated states might interact with halothane. This interaction with channels in both states may explain the greater suppression of INaby halothane at a VHof -80 mV. Furthermore, binding of halothane to channels in the open state might also occur because drug binding can occur rapidly during the first pulse. 
During the experiments with a VHof -110 mV, where all channels were available for opening, the effect of halothane on INawas completely reversible. In contrast, from a VHof -80 mV where approximately 14% of the available channels are inactivated, washout of halothane resulted in only a 50% recovery of Na sup + current. With a V sub H of -80 mV, washout was complete only after application of a conditioning hyperpolarizing pulse to -110 mV. This suggests that if the channel can change its conformational state to the resting state, halothane may be more likely to unbind. However, the delay with which the recovery occurred after the conditioning pulse to -110 mV is puzzling. Recovery is complete only after a 7-min period at -110 mV. If this hyperpolarization of the membrane to -110 mV allows for unbinding of halothane, it is not clear why the process requires a significant length of time. One possibility is that halothane may stabilize the inactivated state of the channel. That this interaction between halothane and the channel is complex is further revealed by the result showing that the rate of recovery from inactivation was not affected during the VHof -110 mV.
Under our experimental conditions, the Na sup + current in guinea pig ventricular myocytes was activated and inactivated within the first 10 ms of the test pulse. In our experiments in which a test pulse duration of 3.5 ms was used, the effects of halothane on Na sup + channel present in the open and resting state are favored because there is little time for an inactivation to occur. Even at those conditions in which only a small fraction of channels are in the inactivated state (VH-110 mV), halothane depressed INa. Thus the channel does not necessarily have to be in the inactivated state to interact with halothane. When these experiments were repeated from a VHof -80 mV, the block induced by halothane was enhanced, as expected, and the block was increased between subsequent test pulses.
From the results of experiments summarized previosusly, a simple model for halothane interaction with the cardiac Na channel is proposed, as shown in the schematics below. A major difference in the halothane effect from VHs of -110 and -80 mV is that from -80 mV, there appears to be an “enhanced” interaction between halothane and the inactivated state of the channel. From -110 mV, this interaction with the inactivated state appears to be minimal. Thus, when using a VHof -110 mV, the interaction between halothane and the channel can be described by the scheme in Figure 10, where C, O, and I denote the closed, open, and inactivated states, respectively. CH denotes a closed state with halothane “bound,” and BH indicates a blocked state with halothane. In this scheme, halothane interacts with the closed and open states of the channel. On the other hand, when using a VHof -80 mV, halothane also interacts with the inactivated state. Thus, from -80 mV, the interaction can be described as in Figure 11. In this scheme, IH denotes an inactivated state stabilized by halothane. This halothane-stabilized inactivated state was incorporated to account for the partial reversibility of the halothane effect from -80 mV and the prolonged recovery period in the presence of a -110 mV prepulse potential. Further, the IH state would account for the slowing of the recovery from inactivation in the presence of halothane from -80 mV. A further marked depression of INawhen using a VHof -80 mV also appears to support the idea that halothane may preferentially bind to the inactivated state.
If the major difference in the interaction between halothane and the Na sup + channel at the two VHs is the interaction with the inactivated state at -80 mV, then removal of channel inactivation at -80 mV would result in the halothane effect observed at -110 mV. The results from the veratridine experiment support this hypothesis. When channel inactivation was removed, the reduction of peak Na sup + current amplitude induced by halothane was similar at both holding potentials and similar to the degree of block obtained when all channels were available for activation. The mechanism of halothane block at -80 mV after removal of channel inactivation is similar to that at -110 mV.
During myocardial ischemia, external K sup + concentrations in the ischemic zone as high as 15 mM have been reported, resulting in depolarization of myocardial cells to between -50 and -60 mV. [7,8] Depolarization is directly responsible for an increase in the number of Na sup + channels in the inactivated state. Fewer channels would thus be available for opening, resulting in a decrease in Vmaxand slowing of cardiac action potential conduction.  Our results show that the magnitude of INablock by halothane is more pronounced when the fraction of channels in the inactivated state is high. These effects would further contribute to a decrease in Vmaxand cardiac conduction in the ischemic myocardium. A previous finding that 0.4 mM halothane reduced an overshoot and the Vmaxin an infarcted but not in control Purkinje fibers supports this hypothesis.  Because of this voltage-dependent effect of halothane on INa, this agent is expected to have a profound affect on Na sup + current in the ischemic cardiac cells under pathophysiologic conditions. Thus electrophysiologic inhomogeneities in cardiac tissue between ischemic and nonischemic fibers may facilitate or block reentrant dysrhythmias, depending on the conduction and refractory properties of the tissue. [21,22]
Although the effects of halothane on cardiac INadepend on the membrane potential, the clinical implication of a partial irreversibility of the effect from a VHof -80 mV is unresolved. Reversal of the halothane effect may require a longer washout period than the one used in this study. On the other hand, the affinity between halothane and the inactivated state of the channel and the rate of transition between the inactivated and the halothane-bound inactivated states may differ at 37 [degree sign] Celsius relative to room temperature. It is also important to note that the mechanisms of interaction of halothane with INainvolves not only different conformational states of the channel but possibly also the second messenger pathways.  Thus the elucidation of the various mechanisms will be necessary to understand the interactions between halothane and the Na sup + channel.