Comparative Effects of Halogenated Inhaled Anesthetics on Voltage-gated Na⁺ Channel Function

Abstract

Background

Inhibition of voltage-gated Na⁺ channels (Na_v) is implicated in the synaptic actions of volatile anesthetics. We studied the effects of the major halogenated inhaled anesthetics (halothane, isoflurane, sevoflurane, enflurane and desflurane) on Na_v1.4, a well characterized pharmacological model for Na_v effects.

Methods

Na⁺ currents (I_Na) from rat Na_v1.4 α-subunits heterologously expressed in Chinese hamster ovary cells were analyzed by whole cell voltage-clamp electrophysiological recording.

Results

Halogenated inhaled anesthetics reversibly inhibited Na_v1.4 in a concentration- and voltage-dependent manner at clinical concentrations. At equi-anesthetic concentrations, peak I_Na was inhibited with a rank order of desflurane > halothane ≈ enflurane > isoflurane ≈ sevoflurane from a physiological holding potential (−80 mV). This suggests that the contribution of Na⁺ channel block to anesthesia might vary in an agent-specific manner. From a hyperpolarized holding potential that minimizes inactivation (−120 mV), peak I_Na was inhibited with a rank order of potency for tonic inhibition of peak I_Na of halothane > isoflurane ≈ sevoflurane > enflurane > desflurane. Desflurane produced the largest negative shift in voltage-dependence of fast inactivation consistent with its more prominent voltage-dependent effects. A comparison between isoflurane and halothane showed that halothane produced greater facilitation of current decay, slowing of recovery from fast inactivation, and use-dependent block than isoflurane.

Conclusions

Five halogenated inhaled anesthetics all inhibit a voltage-gated Na⁺ channel by voltage- and use-dependent mechanisms. Agent-specific differences in efficacy for Na⁺ channel inhibition due to differential state-dependent mechanisms creates pharmacologic diversity that could underlie subtle differences in anesthetic and nonanesthetic actions.

Introduction

Both ligand-gated ion channels, including GABA_A (γ-aminobutyric acid, type A) receptors, glycine receptors, neuronal nicotinic acetylcholine receptors, and N-methyl-D-aspartic acid -type glutamate receptors, as well as voltage-gated ion channels, including Ca²⁺ channels, K⁺ channels and Na⁺ channels, represent promising molecular targets for various general anesthetics.¹ Depression of presynaptic action potential amplitude involving Na⁺ channel blockade has been implicated in inhibition of neurotransmitter release by the potent inhaled (volatile) anesthetics.^2,3 The voltage-gated Na⁺ channel (Na_v) superfamily consists of 9 distinct genes that encode for the channel-forming α-subunit (Na_v1.1–1.9), each with tissue-dependent expression and functions.⁴ The potent inhaled anesthetics inhibit native neuronal Na⁺ channels^5–7 as well as heterologously expressed mammalian Na⁺ channel α-subunits.^8–11 However, the relative potencies and channel gating effects of various inhaled anesthetics have not been compared in detail. Although Na⁺ channel blockade is of enormous therapeutic importance for cardiac dysrhythmias, acute and chronic pain states, seizure disorders and possibly general anesthesia, systemic administration of Na⁺ channel blockers is associated with severe cardiac and central nervous system side-effects.⁴ Effects of anesthetics on central nervous system and peripheral Na_v isoforms are therefore likely to be involved in their anesthetic and some of their agent-specific non-anesthetic effects.

We characterized the effects of five potent inhaled anesthetics on the functionand gating of rat Na_v1.4 α-subunits heterologously expressed in Chinese hamster ovary cells. The skeletal muscle Na⁺ channel isoform Na_v1.4 is expressed at the neuromuscular junction where it regulates muscle excitability.¹² Na_v1.4 function is inhibited by local anesthetic and antidepressant drugs, and provides a well-characterized model for studies of Na⁺ channel pharmacology that is amenable to genetic manipulation for detailed structure-function studies.^13–17 The halogenated alkane halothane and methylethyl ethers isoflurane, sevoflurane, enflurane and desflurane represent the principal volatile anesthetics employed clinically in the modern era. Detailed kinetic characterization of their Na⁺ channel blocking effects is important in identifying common and/or distinct features that might contribute to agent-specific pharmacological profiles. We report here that all five anesthetics inhibit Na_v1.4 at concentrations in the clinical range in proportion to their potencies for producing anesthesia in vivo, which provides additional support for Na⁺ channel blockade as a plausible mechanism of inhaled anesthetic action. In addition, agent-specific differences in their relative potencies and involvement of state-dependent mechanisms could contribute to differences in their central and peripheral pharmacodynamic properties.

Materials and Methods

Cell culture

Chinese hamster ovary cells stably transfected with rat Na_v1.4 α-subunit (a gift from S. Rock Levinson, Ph.D., Professor, Department of Physiology and Biophysics, University of Colorado Health Sciences Center, Denver, Colorado) were cultured in 90% (v/v) DMEM medium, 10% (v/v) fetal bovine serum, 300 μg/ml G418 (Invitrogen, Carlsbad, CA), 100 units/ml penicillin, and 100 μg/ml streptomycin (Biosource, Rockville, MD) under 95% air/5% CO₂ at 37°C. Cells were plated on glass coverslips in 35-mm plastic dishes (Becton Dickinson, Franklin Lakes, NJ) 1–3 days prior to electrophysiological recording.

Electrophysiology

Cells attached to coverslips were transferred to a plastic Petri dish (35×10 mm) on the stage of a Nikon ECLIPSE TE300 inverted microscope (Melville, NY). The culture medium was replaced and cells were superfused at 1.5–2 ml/min with extracellular solution containing (in mM): NaCl 140; KCl 4; CaCl₂ 1.5; MgCl₂ 1.5; HEPES 10; D-glucose 5; pH 7.30 with NaOH. Studies were conducted at room temperature (24±1°C) using conventional whole cell patch-clamp techniques.¹⁸ Patch electrodes (tip diameter <1 μm) were made from borosilicate glass capillaries (Drummond Scientific, Broomall, PA) using a micropipette puller (P-97, Sutter Instruments, Novato, CA) and fire polished (Narishige Microforge, Kyoto, Japan). Electrode tips were coated with SYLGARD (Dow Corning Corporation, Midland, MI) to lower background noise and reduce capacitance; electrode resistance was 2–5 MΩ. The pipet electrode solution contained (in mM): CsF 80; CsCl 40; NaCl 15; HEPES 10; EGTA 10; pH 7.35 with CsOH. Currents were sampled at 10 kHz and filtered at 1–3 kHz using an Axon 200B amplifier, digitized via a Digidata 1321A interface, and analyzed using pClamp 8.2 (Axon/Molecular Devices, Sunnyvale, CA). Capacitance and 60–85% series resistance were compensated and leak current was subtracted using P/4 or P/5 protocols. Cells were held at −80 mV between recordings. Only cells with Na⁺ currents of 0.5–3.5 nA were analyzed in order to minimize increasing series resistance and contributions of endogenous Na⁺ currents (<50 pA) occasionally observed in Chinese hamster ovary cells.¹⁹

Anesthetics

Thymol-free halothane was obtained from Halocarbon Laboratories (River Edge, NJ); isoflurane and sevoflurane were from Abbott Laboratories (Abbott Park, IL), enflurane was from Anaquest Inc. (Liberty Corner, NJ), and desflurane was from Baxter Healthcare Corporation (Deerfield, IL). Anesthetics were diluted from saturated aqueous stock solutions made in extracellular solution (14–16 mM halothane, 10–12 mM isoflurane, 4–6 mM sevoflurane, 10–12 mM enflurane, 8–10 mM desflurane) prepared 12–24 h prior to experiments into airtight glass syringes, and applied locally to attached cells at 50–70 μl/min using an ALA-VM8 pressurized perfusion system (ALA Scientific, Westbury, NY) through a 0.15 mm diameter perfusion pipette positioned 30–40 μm away from patched cells. Concentrations of volatile anesthetics were determined by local sampling of the perfusate at the site of the recording pipette tip and analysis by gas chromatography as described. ⁵

Statistical analysis

IC₅₀ values were calculated by least squares fitting of data to the Hill equation: Y=1/(1+10((logIC₅₀−X)*h)), where Y is the effect, X is measured anesthetic concentration, and h is Hill slope. Activation curves were fitted to a Boltzmann equation of the form G/G_max=1/(1+e(V_1/2a−V)/k), where G/G_max is normalized fractional conductance, G_max is maximum conductance, V_1/2a is voltage for half-maximal activation, and k is the slope factor. Na⁺ conductance (G_Na) was calculated using the equation: G_Na=I_Na/(V_t−V_r), where I_Na is peak Na⁺ current, V_t is test potential and V_r is Na⁺ reversal potential (E_Na=69 mV). Fast inactivation curves were fitted to a Boltzmann equation of the form I/I_max=1/(1+e(V_1/2in−V)/k), where I/I_max is normalized current, I_max is maximum current, V_1/2in is voltage of half maximal inactivation, and k is slope factor. I_Na current decay was analyzed by fitting the decay phase of the current trace between 90% and 10% of maximal I_Na to the mono-exponential equation I_Na=A·exp(−t/τ_in)+C, where A is maximal I_Na amplitude, C is plateau I_Na, t is time, and τ_in is time constant of current decay. Channel recovery from fast inactivation was fitted to the mono-exponential function Y=A*(1−exp(−τ_r*X)), where Y is fractional current recovery constrained to 1.0 at infinity time, A is normalized control amplitude, X is recovery time, and τ_r is time constant of recovery, and the goodness of fit compared to that of a bi-exponential function. The effects of anesthetics were compared to control using sum-of-squares F-test between curve fits of mean data. The time course of use-dependent decay of normalized I_Na was analyzed by fitting to the mono-exponential equation I_Na=exp(− τ_use·n)+C, where n is pulse number, C is plateau I_Na, and τ_use is time constant of use-dependent decay. Data were analyzed using pClamp 8.2 (Axon/Molecular Devices), Prism 4.0 (Graph-Pad Software Inc., San Diego, CA) and SigmaPlot 6.0 (SPSS Science Software Inc., Chicago, IL). Curve fits were compared by sum-of-squares F-test. Statistical significance was assessed by analysis of variance with Newman-Keuls post hoc test or paired or unpaired t-tests, as appropriate; p<0.05 was considered statistically significant.

Results

Inhibition of peak I_Na

Average peak Na⁺ current (I_Na) in Chinese hamster ovary cells transfected with the Na_v1.4 α-subunit was −3.0±0.2 nA (n=11) from a holding potential of −120 mV. Peak I_Na was rapidly (onset<1.5 min) and reversibly inhibited by all five inhaled anesthetics tested (fig. 1), and by the specific Na⁺ channel blocker tetrodotoxin (data not shown). Inhibition was greater from the more physiological holding potential of −80 mV than from the hyperpolarized potential of −120 mV (fig. 2), indicative of significant voltage-dependent inhibition. At aqueous concentrations equivalent to ~1 Minimum alveolar concentration (MAC) for rat (0.35 mM for halothane and isoflurane, 0.46 mM for sevoflurane, 0.75 mM for enflurane, and 0.80 mM for desflurane),²⁰ desflurane showed the greatest inhibition of peak I_Na from a holding potential of −80 mV. The rank order of inhibition was desflurane (57±6.2% @ 0.83±0.06 mM, n=4) > halothane (32±3.5% @ 0.42±0.05 mM, n=6) enflurane (32±6.7% @ 0.82±0.06 mM, n=5) > isoflurane (19±1.9% @ 0.46±0.04 mM, n=4) sevoflurane (17±3.3% @ 0.44±0.04 mM, n=4; mean±SEM). The degree of voltage-dependent inhibition (difference between efficacy at −80 mV vs. −120 mV) varied between anesthetics, and was greatest for desflurane and least for halothane (fig. 2).

Fig. 1.

Inhibition of Na_v1.4 by equipotent concentrations of various inhaled anesthetics. Na⁺ currents (I_Na) were recorded from a holding potential of −80 mV by 25 ms test steps to V_max (−10 or −20 mV) as shown in the inset. The effects of halothane (A, 0.40 mM; 1.1 MAC), isoflurane (B, 0.42 mM; 1.2 MAC) and sevoflurane (C, 0.46 mM; 1.0 MAC), enflurane (D, 0.81 mM; 1.1 MAC) and desflurane E, (0.85 mM; 1.1 MAC) at ~1 MAC are shown in these representative traces (summary data are given in the Results). The time-course of I_Na inhibition expressed as fractional I_Na (I_Na/I_Na control) during application of isoflurane (0.43 mM, 1.2 MAC) or halothane (0.39 mM, 1.1 MAC) for 1.5 min is shown in F. I_Na was repetitively activated from a holding potential of −120 mV by 25 ms test pulses to −10 mV at 0.5 sec intervals.

Fig. 2.

Voltage-dependent inhibition of Na_v1.4 by inhaled anesthetics. Equipotent concentrations (~1 MAC) of inhaled anesthetics differentially inhibited I_Na from a holding potential of −80 mV (open bars) or −120 mV (filled bars). The measured concentrations of halothane (Halo), isoflurane (Iso), sevoflurane (Sevo), enflurane (Enf), and desflurane (Des) were 0.42±0.05 mM (1.2 MAC), 0.46±0.03 mM (1.3 MAC), 0.44± 0.03 mM (1.0 MAC), 0.82±0.04 mM (1.1 MAC), and 0.83±0.03 mM (1.0 MAC), respectively, from a holding potential of −80 mV; and were 0.38±0.05 mM (1.1 MAC), 0.41±0.04 mM (1.1 MAC), 0.45±0.05 mM (1.0 MAC), 0.80±0.05 mM (1.1 MAC), and 0.83±0.04 mM (1.0 MAC), respectively, from a holding potential of −120 mV. Data are expressed as mean±SEM (n=4–12). ** P < 0.01 by unpaired t-test.

Voltage-gated Na⁺ channels have at least three distinct conformational states: resting, open, and inactivated.¹³ The potency of each anesthetic for tonic inhibition was investigated using a holding potential of −120 mV to maintain channels in the resting state allowing assessment of resting channel block with minimal interference from voltage-dependent inactivation. All five anesthetics exhibited concentration-dependent inhibition of peak I_Na with IC₅₀ values in the millimolar range and Hill slopes of ~2, except for halothane which had a Hill slope of ~1 (fig. 3); this suggests the possibility of two sites of interaction with Na_v1.4 for the ethers vs. a single site of interaction for the alkane.

Fig. 3.

Concentration dependence of Na_v1.4 inhibition by inhaled anesthetics. Normalized peak I_Na values from a holding potential of −120 mV were fitted to the Hill equation to yield IC₅₀ values (±SE) and Hill slopes. IC₅₀ values differed from each other by sum-of-squares F-test (P < 0.05). □, Halothane; ▲, Isoflurane; ▽, Sevoflurane; ◆, Enflurane; ○, Desflurane.

Effects on channel gating

None of the anesthetics tested altered the current-voltage relationship or reversal potential for I_Na (fig. 4; data not shown). At concentrations equivalent to ~1 MAC, all five anesthetics produced no significant shift in the voltage dependence of activation (fig. 5) with minor effects on slope factors (data not shown). The voltage dependence of fast inactivation was determined for the prototypical anesthetic isoflurane, and for desflurane and halothane which exhibited extremes in voltage sensitivity (fig. 2). Representative current traces, which reflect channel availability at various holding potentials, obtained using a protocol designed to minimize the influence of slow inactivation are shown in figure 6A. Isoflurane, halothane and desflurane strongly enhanced inactivation in a concentration-dependent manner. Desflurane produced a greater negative shift in V_1/2in than isoflurane or halothane as seen in curve fits of the mean data (fig. 6B,C). At concentrations of ~1 MAC, isoflurane, halothane and desflurane shifted V_1/2in by −7 mV, −9 mV and −13 mV, respectively (fig. 6B). Similar effects were evident when the data were analyzed by calculating the mean values of V_1/2in derived from curve fits of the individual data sets (table 1). Slope factors, which reflect the voltage sensitivity of the inactivation gate,²¹ were not significantly affected by isoflurane and halothane, but were slightly increased by desflurane (table 1). Macroscopic current inactivation was examined by fitting the rate of decay of current elicited by depolarization from −120 mV to V_max to a mono-exponential equation. Time constants of current decay (τ_in) were reduced by desflurane > halothane > isoflurane (table 2). The effects of isoflurane and halothane on recovery of I_Na from fast inactivation were evaluated by a two-pulse protocol with varying time intervals (fig. 7). Both anesthetics slowed recovery from inactivation by increasing the time constant of recovery (τ_r, ms) derived from mono-exponential fits of the fractional current (fig. 7).

Fig. 4.

Effects of isoflurane and halothane on Na_v1.4 current-voltage relationship. A. Isoflurane (left) or halothane (right) at concentrations equivalent to ~2 MAC significantly inhibited I_Na from a holding potential of −120 mV. B. Voltage of peak I_Na activation (V_max) was −10 mV; neither anesthetic affected voltage of peak current activation or reversal potential. Mean concentrations of isoflurane was 0.84±0.08 mM (2.4 MAC) and of halothane was 0.81±0.06 mM (2.3 MAC) (n=5–8). Comparable results were obtained for desflurane, enflurane, and sevoflurane (data not shown). *** P < 0.001, ** P < 0.01 vs. control by paired t-test.

Fig. 5.

Effects of volatile anesthetics on voltage-dependence of Na_v1.4 activation. Normalized conductance data (mean±SEM) were fitted to a Boltzmann equation to yield voltage of 50% maximal activation (V_1/2a) and slope factor. Holding potential was −120 mV (n=5–12). There were no significant shifts in V_1/2a or slope factor (P > 0.05 by t-test). See Fig. 2 legend for abbreviations.

Fig. 6.

Effects of isoflurane, halothane and desflurane on voltage dependence of Na_v1.4 fast inactivation. A. Representative traces show inhibition of I_Na by isoflurane (left), halothane (middle) and desflurane (right) using a fast inactivation protocol (inset) involving a conditioning pulse of 30 ms followed by a test pulse of 25 ms to minimize slow inactivation. Normalized data were fitted to the Boltzmann equation to yield voltage of 50% inactivation (V_1/2in) and slope factor for ~1 MAC (B) or ~2 MAC (C). Each anesthetic significantly shifted the V_1/2in in the negative direction as determined by sum-of-squares F-test comparison between curve fits of mean data (P < 0.05). Parameters derived from analysis of independent curve fits are presented in Table 1. Anesthetic concentrations were 0.46±0.09 mM and 0.82±0.07 mM for isoflurane; 0.40±0.06 mM and 0.77±0.10 mM for halothane; and 0.82±0.06 mM and 1.61±0.07 mM for desflurane. Data expressed as mean±SEM, n=5–7.

Table 1.

Inhaled Anesthetic Effects on Na_v1.4 Inactivation.

~1 MAC	V1/2in, mV	k	n
Control	−49.1 ± 1.4	6.5 ± 0.2	4
Isoflurane (0.46 ± 0.09 mM)	−55.4 ± 2.6	6.9 ± 0.1	4
Control	−48.8 ± 2.5	7.0 ± 0.5	7
Halothane (0.40 ± 0.06 mM)	−57.8 ± 2.5‡	7.2 ± 0.4	7
Control	−57.2 ± 1.1	6.6 ± 0.1	5
Desflurane (0.82 ± 0.06 mM)	−69.7 ± 3.0†§||	7.4 ± 0.3*	5
~2 MAC
Control	−53.2 ± 2.3	7.1 ± 0.3	5
Isoflurane (0.82 ± 0.07 mM)	−63.3 ± 2.1*	8.4 ± 0.9	5
Control	−54.1 ± 2.2	7.3 ± 0.4	5
Halothane (0.77 ± 0.10 mM)	−64.9 ± 2.8‡	7.9 ± 0.6	5
Control	−58.7 ± 1.2	6.7 ± 0.1	4
Desflurane (1.61 ± 0.07 mM)	−83.2 ± 1.6‡§||	7.5 ± 0.1*	4

Data for each experiment were fitted to a standard Boltzmann equation, and V_1/2in and slope values for each experiment were averaged and displayed as mean ± SEM.

^*P < 0.05,

^†P <0.01,

^‡P < 0.001 vs. respective control by paired t test.

^§P < 0.05 vs. isoflurane,

^||P < 0.05 vs. halothane by one-way analysis of variance with Newman-Keuls post hoc test. k = slope factor; MAC = minimum alveolar concentration; V1/2in = voltage of half-maximal inactivation.

Table 2.

Inhaled anesthetic effects on Na_v1.4 current decay.

	τin, ms
	Control	Anesthetic	Concentration, mM	n
Isoflurane	0.54 ± 0.08	0.47 ± 0.08*	0.46 ± 0.05	8
	0.52 ± 0.09	0.43 ± 0.08*	0.85 ± 0.04	6
Halothane	0.51 ± 0.07	0.42 ± 0.04*	0.43 ± 0.03	5
	0.55 ± 0.07	0.39 ± 0.06†	0.81 ± 0.04	5
Desflurane	0.55 ± 0.10	0.40 ± 0.05*‡	0.80 ± 0.06	7
	0.54 ± 0.09	0.34 ± 0.08†‡§	1.65 ± 0.14	5

Values are mean ± SEM.

^*P < 0.01,

^†P < 0.001 vs. respective control by unpaired t test.

^‡P < 0.05 vs. isoflurane,

^§P < 0.05 vs. halothane by one-way analysis of variance with Newman-Keuls post hoc test. τ_in = time constant of current decay calculated for a prepulse potential of −120 mV.

Fig. 7.

Effects of isoflurane and halothane on recovery of Na_v1.4 from fast inactivation. The protocol, shown in the upper right inset, involved 12 depolarizing test steps at various recovery times (t) in 2.5 ms intervals. Representative current traces obtained for the effects of isoflurane (~2.3 MAC) and halothane (~2.2 MAC) are shown on the right. The time course of channel recovery from fast inactivation was best fitted by a mono-exponential function in all cases to yield a recovery time constant (τ_r). The rate of recovery, expressed as current normalized to initial control current, was slowed by isoflurane (A) and halothane (B). The recovery time constant was greater for halothane than for isoflurane at the higher concentrations as determined by sum-of-squares F-test between curve fits of mean data (P < 0.001). Mean isoflurane concentrations were 0.44±0.06 mM and 0.82±0.08 mM; mean halothane concentrations were 0.41±0.07 mM and 0.78±0.10 mM. Data are expressed as mean±SEM, n=5–8. *** P < 0.001 vs. control.

Use-dependent block

Use-dependent block of Na_v1.4 was evident as a reduction in normalized I_Na relative to the peak of the first pulse evaluated in a series of rapid depolarizing pulses (fig. 8). In the absence of anesthetic, repetitive pulses produced only small reductions in peak I_Na. Both halothane and isoflurane at ~2 MAC reduced the time constant of use-dependent decay (τ_use). Halothane produced a greater reduction in steady-state normalized I_Na amplitude than isoflurane (P < 0.05 by paired t-test, n=3). These results are consistent with contributions of open channel block and/or enhanced inactivation to inhibition of Na_v1.4.

Fig. 8.

Enhanced use-dependent block of Na_v1.4 by isoflurane and halothane. A. Representative current traces showing isoflurane (left) and halothane (right; solid lines) enhancement of use-dependent block of I_Na compared to control (dotted lines) from a holding potential of −120 mV. B. Peak currents were normalized to the current of the first pulse (mean±SEM; n=3), plotted against pulse number, and fitted to a mono-exponential function. Halothane (0.82±0.06 mM) reduced the time constant of use-dependent decay (τ_use) from 7.3 to 2.0 pulses, and isoflurane (0.85±0.08 mM) reduced τ_use from 3.3±0.02 to 1.6±0.02 pulses (mean, n=3). Halothane produced a significantly greater reduction in the plateau I_Na amplitude (0.94±0.02 to 0.63±0.02) than isoflurane (0.90±0.01 to 0.76±0.01; P < 0.05 by paired t-test, n=3). Repetitive pulse protocol: 25 ms, 10 Hz test pulses from holding potential of −120 mV to peak activation voltage.

Discussion

We compared the actions of five potent halogenated inhaled anesthetics on a single Na⁺ channel isoform (Na_v1.4) expressed in a uniform mammalian cellular environment to enhance detection of possible agent specific effects on state-dependent inhibition. All five of these clinically used inhaled anesthetics, with representatives from both alkane and ether subclasses, inhibited currents conducted by the α-subunit of the Na_v1.4 voltage-gated Na⁺ channel isoform at clinically relevant concentrations consistent with a role for blockade of Na_v in anesthetic immobilization.¹ There were differences between agents in their potencies for inhibition of Na_v1.4 relative to their anesthetic potencies, as well as differences in the voltage-dependence of their inhibition. For example, at equi-anesthetic concentrations desflurane was the most effective inhibitor of peak I_Na from a near physiological holding potential of −80 mV while halothane was most effective from a hyperpolarized holding potential of −120 mV. Thus members of the same drug class have agent-specific differences in their effects on a single target with potential pharmacological implications. A clinical implication of these findings is that inhibition of Na_v1.4 could contribute to skeletal muscle relaxing effects of anesthetics given the high density of Na_v1.4 at the neuromuscular junction.¹² Indeed the greater inhibition of Na_v1.4 by desflurane at 1 MAC correlates with its relatively greater enhancement of nondepolarizing neuromuscular blocking drug potency during anesthesia in vivo in human subjects.²²

Inhibition of Na_v1.4 from a hyperpolarized holding potential is consistent with anesthetic block of the closed resting state.²¹ Our results are comparable to those reported for the cardiac isoform Na_v1.5, for which equi-anesthetic concentrations of halothane are more potent than isoflurane in tonic blockade of human⁹ and guinea pig²³ cardiac I_Na. Isoflurane, halothane and desflurane, which were analyzed in more detail, exhibited state-dependent block and a negative shift in the voltage dependence of inactivation, consistent with enhancement of inactivation at physiological holding potentials. State-dependent block has been reported previously for isoflurane effects on I_Na in rat neurohypophysial nerve terminals,⁷ guinea pig cardiomyocytes,²³ and heterologously expressed Na_v1.2, Na_v1.4, Na_v1.5 and Na_v1.6.^8–11 Moreover, isoflurane and halothane affected channel gating evident as accelerated current decay and use-dependent block (halothane > isoflurane). The similarity of these effects of inhaled anesthetics to the effects of local anesthetics, antidepressants and anticonvulsants on Na_v1.2 and Na_v1.4^13–17 currents suggests conserved or overlapping drug binding sites and/or allosteric conformational mechanisms for these chemically diverse Na⁺ channel antagonists. The relative contributions of open state block and enhanced fast and/or slow inactivation to use-dependent block by inhaled anesthetics, which differs between various Na⁺ channel blockers, should be resolvable by examining anesthetic effects on fast inactivation-deficient Na⁺ channels.

Inhaled anesthetics are known to inhibit various isoforms of Na_v α-subunits heterologously expressed in Chinese hamster ovary cells (rat Na_v1.2, Na_v1.4 and Na_v1.5),¹¹ human embryonic kidney cells (HEK 293, human Na_v1.5),⁹ and Xenopus oocytes (rat Na_v1.2 and 1.6, human Na_v1.4).¹⁰ Small differences in potencies reported between various studies probably result from differences in isoform sensitivity, species, expression systems, β-subunit co-expression, recording conditions, stimulation protocols, anesthetic concentration determinations, etc. Isoflurane at clinically relevant concentrations inhibits rat neuronal (Na_v1.2), skeletal muscle (Na_v1.4), and cardiac muscle (Na_v1.5) voltage-gated Na⁺ channel α-subunits studied under identical conditions with isoform-dependent differences in state-dependent block.¹¹ Significantly lower IC₅₀ values for isoflurane were observed at more physiological holding potentials¹¹ due to marked voltage-dependent effects on channel gating compared to the hyperpolarized potential used to characterize tonic block in the current study. The IC₅₀ for inhibition of Na_v1.4 by isoflurane reported previously for a holding potential of −100 mV (IC₅₀=0.99 mM)¹¹ compares well with the value obtained in the present study for a holding potential of −120 mV (IC₅₀=1.16 mM). Rat Na_v1.8 in Xenopus oocytes has been reported to be insensitive to isoflurane,¹⁰ but recent evidence indicates that rat Na_v1.8 expressed in a mammalian neuroblastoma cell line is inhibited by isoflurane at concentrations comparable to those effective on other isoforms (unpublished data, 2008, Karl F. Herold, M.D. and Hugh C. Hemmings, M.D., Ph.D., New York NY). Thus all mammalian Na_v isoforms tested so far are susceptible to inhibition by the prototypical inhaled anesthetic isoflurane with minor isoform-specific differences in relative potency and mechanism.

Human Na_v1.4 heterologously expressed in Xenopus oocytes is inhibited by isoflurane and halothane,¹⁰ whereas rat Na_v1.4 in the same expression system was reported to be insensitive to halothane unless co-expressed with protein kinase C (PKC).²⁴ Our results indicate that rat Na_v1.4 expressed in a mammalian cell line is inhibited by multiple inhaled anesthetics in the absence of over-expression or pharmacological activation of PKC indicating that PKC activation is apparently not required for inhibition. A requirement for activation of endogenous PKC, which can be activated by halogenated inhaled anesthetics,²⁵ cannot be excluded however. Attempts to test this possibility by inhibition of endogenous PKC using small molecule PKC inhibitors have been unsuccessful since the PKC inhibitors themselves inhibit Na⁺ channels.²⁶

Inhaled anesthetics negatively shift the voltage dependence of I_Na fast inactivation. Similar shifts in inactivation of I_Na are produced in ventricular cardiomyocytes by isoflurane and halothane,⁹ and in rat neurohypophysial nerve terminals⁷ and heterologously expressed Na_v isoforms by isoflurane.^10,11 Negative shifts in the voltage dependence of fast inactivation suggest greater anesthetic binding affinity and selective stabilization of inactivated states. The large negative shift in V_1/2in by desflurane contributes to its greater potency compared to isoflurane and halothane for inhibition of Na_v1.4 from the more positive (physiological) holding potential of −80 mV vs. −120 mV. Preferential anesthetic interaction with the nonconducting inactivated state of Na_v1.4 is also consistent with anesthetic slowing of recovery from fast inactivation. The slightly greater slowing effect of halothane compared to isoflurane is consistent with a greater shift in V_1/2in and hence stronger interaction with the fast inactivated state for halothane.

Enhanced inactivation is critical to inhibition of Na_v1.4 by inhaled anesthetics at more positive membrane potentials. This has classically been attributed to fast inactivation, but recent evidence implicates slow inactivation in activity-dependent attenuation of Na_v currents.⁴ The possible role of slow inactivation in the Na⁺ channel blocking effects of inhaled anesthetics is not clear, but is an important area for future investigation that will be facilitated using mutations in Na_v1.4 that enhance or remove inactivation.²⁷ Possible mechanisms underlying state-dependent effects include facilitated transitions from the closed to inactivated state (tonic block), open to inactivated state (enhanced inactivation), and/or stabilization of inactivated states (delayed recovery). Small differences between anesthetics in their state-dependent effects on various Na_v isoforms¹¹ could underlie anesthetic- and isoform-specific differences in pharmacological profiles of specific anesthetics in vivo, e.g., differential effects on brain, heart and skeletal muscle.

All five inhaled anesthetics tested produced insignificant shifts in the voltage dependence of Na_v1.4 activation, similar to the findings of Stadnicka et al.,⁹ but in contrast to those of Weigt et al.²³ who found small negative shifts for Na_v1.5. These findings suggest isoform-selective interactions between volatile anesthetics and the activation process similar to those observed for the local anesthetic lidocaine.²⁸ Another class of anesthetics, the n-alkanols, also inhibits voltage-gated Na⁺ channels at anesthetic concentrations, but with somewhat distinct mechanisms from those of inhaled anesthetics that involve primarily open channel block with relatively small effects on both activation and inactivation.²⁹ Thus the n-alkanols and inhaled anesthetics both inhibit Na⁺ channels, but differ in the relative involvement of open channel block and activation (greater for the alkanols) versus inactivation mechanisms (greater for inhaled anesthetics).

Both halothane and isoflurane exhibited use-dependent block with repetitive stimuli. The fraction of open versus inactivated channels increases during fast repetitive depolarizations, so the presence of use-dependent block suggests a possible role for open-channel block and/or slow inactivation mechanisms by both anesthetics.^17,21 Halothane was more efficacious than isoflurane in inhibiting normalized current amplitude with repetitive stimuli consistent with its greater tonic I_Na blocking effect. Open-channel block is particularly important in pathological conditions such as myotonia and periodic paralysis that involve Na_v1.4 inactivation gating defects,³⁰ inflammatory and neuropathic pain states that involve repetitive activation of Na_v1.7 and Na_v1.8,^31,32 and ischemia which leads to resting membrane depolarization.³³

Accessory subunits can have important effects on the pharmacological and gating properties of voltage-gated Na⁺ channels that must be considered in pharmacological studies, although α-subunit expression is sufficient to mimic native Na⁺ channel gating properties.⁴ Modulation of Na_v function by β–subunits depends on both the α subunit isoform and the cell type used for expression. Co-expression of the β1-subunit has no effect on inhibition by isoflurane of Na_v1.2, Na_v1.4, Na_v1.6 or Na_v1.8 α-subunits expressed in Xenopus oocytes.¹⁰ In mammalian expression systems, β1-subunit co-expression has no major effects on local anesthetic sensitivity, current kinetics, or activation and inactivation properties of tetrodotoxin-sensitive currents in ND7/23 cells,³⁴ but produces positive shifts of channel activation and inactivation of Na_v1.2 in tsA-201 cells³⁵ and positive shifts of inactivation but no change in cocaine affinity of Na_v1.4 and Na_v1.5 in human embryonic kidney 293t cells³⁶ Although we have not ruled out possible effects of β–subunit co-expression on inhaled anesthetic sensitivity of Na_v1.4 in Chinese hamster ovary cells, taken together these findings make a major effect unlikely.

The role of voltage-gated Na⁺ channels in the mechanisms of inhaled anesthetics is an important and unresolved question, but a number of factors impede its resolution.¹ Correlations between in vitro effects on Na⁺ currents and anesthetic potencies in vivo are limited by our ignorance regarding the specific cells, networks and molecular targets involved in the behavioral effects of inhaled anesthetics (immobilization in the case of MAC). Thus we are unable to define the degree of Na⁺ channel inhibition critical for an anesthetic effect that must be taken into consideration when evaluating correlations between potencies measured in vitro and in vivo. Interestingly, experiments with local anesthetics suggest that relatively small degrees of Na_v blockade (10–20% inhibition of peak current amplitude) can have profound effects on neuronal firing rate.³⁷ Moreover, determination of anesthetic effects on ion channels in vitro is subject to numerous experimental variables including the species and isoform of the channel, expression system used, accessory subunits, modulation by cellular signaling pathways, temperature, experimental conditions including holding potentials and stimulus protocols, etc. These and other factors can have profound effects on channel function and pharmacological sensitivity. Given these reservations, the observation that all five inhaled anesthetics tested inhibit Na_v1.4 supports, but does not prove, an important role for Na_v inhibition in anesthesia. Additional support for this hypothesis is provided by the recent observation that intrathecal administration of the Na_v agonist veratridine increases MAC in rats.³⁸

In summary, halogenated inhaled anesthetics all inhibit heterologously expressed Na_v1.4 at clinical concentrations by state-dependent mechanisms. These findings support inhibition of Na_v as a common mechanism for inhaled anesthetic action. Small agent-specific differences in relative potency and gating effects are consistent with subtle inter-agent variability in pharmacodynamic profiles, such as skeletal muscle relaxant effects. Agent-specific differences in potency for Na_v1.4 inhibition at normal resting membrane potential were determined primarily by differences in state-dependent block reflected in effects on inactivation gating. These gating effects of inhaled anesthetics are remarkably similar to those of local anesthetics, and suggest the possibility of overlapping binding sites,^13–17 an interesting hypothesis that can now be tested by detailed structure-function studies in Na_v1.4 using mutations that affect gating mechanisms and local anesthetic sensitivity.