Comparative Effects of Halogenated Inhaled Anesthetics on Voltage-Gated Na؉ Channel Function Wei Ouyang, Ph.D.,* Karl F

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Anesthesiology 2009; 110:582–90 Copyright © 2009, the American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins, Inc. Comparative Effects of Halogenated Inhaled Anesthetics on Voltage-gated Na؉ Channel Function Wei Ouyang, Ph.D.,* Karl F. Herold, M.D., Ph.D.,† Hugh C. Hemmings, Jr., M.D., Ph.D.‡

؉ Background: Inhibition of voltage-gated Na channels (Nav) neurotransmitter release by the potent inhaled (volatile) is implicated in the synaptic actions of volatile anesthetics. We 2,3 ϩ anesthetics. The voltage-gated Na channel (Nav)su- studied the effects of the major halogenated inhaled anesthetics perfamily consists of 9 distinct genes that encode for the (halothane, isoflurane, sevoflurane, enflurane, and desflurane) ␣ on Na 1.4, a well-characterized pharmacological model for Na channel-forming -subunit (Nav1.1–1.9), each with tis- v v 4 effects. sue-dependent expression and functions. The potent ؉ ␣ ϩ Methods: Na currents (INa) from rat Nav1.4 -subunits heter- inhaled anesthetics inhibit native neuronal Na chan- Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/110/3/582/367953/0000542-200903000-00024.pdf by guest on 29 September 2021 ologously expressed in Chinese hamster ovary cells were analyzed nels5–7 as well as heterologously expressed mammalian by whole cell voltage-clamp electrophysiological recording. Naϩ channel ␣-subunits.8–11 However, the relative po- Results: Halogenated inhaled anesthetics reversibly inhibited tencies and channel-gating effects of various inhaled an- Nav1.4 in a concentration- and voltage-dependent manner at clinical concentrations. At equianesthetic concentrations, peak esthetics have not been compared in detail. Although Ϸ ϩ INa was inhibited with a rank order of desflurane > halothane Na channel blockade is of enormous therapeutic im- enflurane > isoflurane Ϸ sevoflurane from a physiologic hold- portance for cardiac dysrhythmias, acute and chronic ing potential (–80 mV). This suggests that the contribution of -؉ pain states, seizure disorders, and possibly general anes Na channel block to anesthesia might vary in an agent-specific ϩ manner. From a hyperpolarized holding potential that mini- thesia, systemic administration of Na channel blockers is associated with severe cardiac and central nervous mizes inactivation (–120 mV), peak INa was inhibited with a 4 rank order of potency for tonic inhibition of peak INa of halo- system side effects. Effects of anesthetics on central thane > isoflurane Ϸ sevoflurane > enflurane > desflurane. nervous system and peripheral Nav isoforms are there- Desflurane produced the largest negative shift in voltage-depen- fore likely to be involved in their anesthetic and some of dence of fast inactivation consistent with its more prominent voltage-dependent effects. A comparison between isoflurane their agent-specific nonanesthetic effects. and halothane showed that halothane produced greater facili- We characterized the effects of five potent inhaled tation of current decay, slowing of recovery from fast inactiva- anesthetics on the function and gating of rat Nav1.4 tion, and use-dependent block than isoflurane. ␣-subunits heterologously expressed in Chinese hamster Conclusions: Five halogenated inhaled anesthetics all inhibit ovary cells. The skeletal muscle Naϩ channel isoform a voltage-gated Na؉ channel by voltage- and use-dependent ,؉ Nav1.4 is expressed at the neuromuscular junction mechanisms. Agent-specific differences in efficacy for Na 12 channel inhibition due to differential state-dependent mecha- where it regulates muscle excitability. Nav1.4 function nisms creates pharmacologic diversity that could underlie sub- is inhibited by local anesthetic and antidepressant drugs, tle differences in anesthetic and nonanesthetic actions. and it provides a well-characterized model for studies of Naϩ channel pharmacology that is amenable to genetic 13–17 ␥ manipulation for detailed structure-function studies. BOTH ligand-gated ion channels, including GABAA ( - aminobutyric acid, type A) receptors, glycine receptors, The halogenated alkane halothane and methylethyl neuronal nicotinic acetylcholine receptors, and N-meth- ethers isoflurane, sevoflurane, enflurane, and desflurane represent the principal volatile anesthetics employed yl-D-aspartic acid–type glutamate receptors, as well as 2ϩ ϩ clinically in the modern era. Detailed kinetic character- voltage-gated ion channels, including Ca channels, K ϩ channels, and Naϩ channels, represent promising molec- ization of their Na channel blocking effects is impor- ular targets for various general anesthetics.1 Depression tant for identifying common and/or distinct features that of presynaptic action potential amplitude involving Naϩ might contribute to agent-specific pharmacological pro- channel blockade has been implicated in inhibition of files. We report here that all five anesthetics inhibit Nav1.4 at concentrations in the clinical range in propor- tion to their potencies for producing anesthesia in vivo, * Research Associate, ‡ Professor and Vice Chair of Research, Departments of ϩ Anesthesiology and Pharmacology, Weill Cornell Medical College, New York, which provides additional support for Na channel New York; † Postdoctoral Associate, Department of Anesthesiology, Weill Cor- blockade as a plausible mechanism of inhaled anesthetic nell Medical College, and Department of Anesthesiology and Intensive Care, Campus Mitte, Charite´ University Medicine Berlin, Berlin, Germany. action. In addition, agent-specific differences in their Received from the Department of Anesthesiology, Weill Cornell Medical Col- relative potencies and involvement of state-dependent lege, New York, New York. Submitted for publication July 8, 2008. Accepted for mechanisms could contribute to differences in their cen- publication November 18, 2008. Supported by grant GM 58055 from the National Institutes of Health, Bethesda, Maryland, (to Dr. Hemmings), and by fellowship tral and peripheral pharmacodynamic properties. HE4554/5-1 from the Deutsche Forschungsgemeinschaft, Bonn, Germany (to Dr. Herold). Drs. Ouyang and Herold contributed equally to this study. Address correspondence to Dr. Hemmings: Department of Anesthesiology, Materials and Methods Box 50, LC-203, Weill Cornell Medical College, 1300 York Avenue, New York, New York 10065. [email protected]. Information on purchasing re- Cell Culture prints may be found at www.anesthesiology.org or on the masthead page at the Chinese hamster ovary cells stably transfected with rat beginning of this issue. ANESTHESIOLOGY’s articles are made freely accessible to all ␣ readers, for personal use only, 6 months from the cover date of the issue. Nav1.4 -subunit (a gift from S. Rock Levinson, Ph.D.,

Anesthesiology, V 110, No 3, Mar 2009 582 INHALED ANESTHETIC EFFECTS ON NAV1.4 583

Professor, Department of Physiology and Biophysics, Uni- pette (diameter, 0.15 mm) positioned 30–40 ␮m away versity of Colorado Health Sciences Center, Denver, Colo- from patched cells. Concentrations of volatile anesthet- rado) were cultured in 90% (v/v) Dulbecco’s Modiﬁed ics were determined by local sampling of the perfusate at Eagle Medium, 10% (v/v) fetal bovine serum, 300 ␮g/ml the site of the recording pipette tip and analysis by gas G418 (Invitrogen, Carlsbad, CA), 100 units/ml penicillin, chromatography as described.5 and 100 ␮g/ml streptomycin (Biosource, Rockville, MD) under 95% air/5% CO2 at 37°C. Cells were plated on glass Statistical Analysis coverslips in 35-mm plastic dishes (Becton Dickinson, IC50 values were calculated by least squares ﬁtting of data to Franklin Lakes, NJ) 1–3 days before electrophysiological ϭ ϩ Ϫ ϫ the Hill equation: Y 1/(1 10((logIC50 X) h)), where recording. Y is the effect, X is measured anesthetic concentration, and h

is Hill slope. Activation curves were fitted to a Boltzmann Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/110/3/582/367953/0000542-200903000-00024.pdf by guest on 29 September 2021 Electrophysiology ϭ ϩ equation of the form G/Gmax 1/(1 e(V1/2a – V)/k), where Cells attached to coverslips were transferred to a plas- G/G is normalized fractional conductance, G is maxi- tic Petri dish (35 ϫ 10 mm) on the stage of a Nikon max max mum conductance, V1/2a is voltage for half-maximal activa- ECLIPSE TE300 inverted microscope (Melville, NY). The ϩ tion, and k is the slope factor. Na conductance (GNa) was culture medium was replaced, and cells were superfused ϭ calculated using the equation: GNa INa/(Vt –Vr), where INa at 1.5–2 ml/min with extracellular solution containing ϩ ϩ is peak Na current, Vt is test potential, and Vr is Na reversal (in mM): NaCl 140; KCl 4; CaCl2 1.5; MgCl2 1.5; HEPES ϭ potential (ENa 69 mV). Fast inactivation curves were fitted 10; D-glucose 5; pH 7.30 with NaOH. Studies were con- to a Boltzmann equation of the form I/I ϭ 1/(1 ϩ e(V ducted at room temperature (24 Ϯ 1°C) using conven- max 1/2in 18 – V)/k), where I/I max is normalized current, I max is maximum tional whole cell patch-clamp techniques. Patch elec- current, V is voltage of half maximal inactivation, and k is trodes (tip diameter Ͻ 1 ␮m) were made from borosilicate 1/2in slope factor. INa current decay was analyzed by fitting the glass capillaries (Drummond Scientific, Broomall, PA) using decay phase of the current trace between 90% and 10% of a micropipette puller (P-97; Sutter Instruments, Novato, ϭ maximal INa to the monoexponential equation INa A· CA) and fire polished (Narishige Microforge, Kyoto, Japan). Ϫ ␶ ϩ exp( t/ in) C, where A is maximal INa amplitude, C is Electrode tips were coated with SYLGARD (Dow Corning ␶ plateau INa, t is time, and in is time constant of current decay. Corporation, Midland, MI) to lower background noise and Channel recovery from fast inactivation was fitted to the reduce capacitance; electrode resistance was 2–5 M⍀. The ϭ ϫ Ϫ␶ ϫ monoexponential function Y A (1 – exp( r X)), pipette electrode solution contained (in mM): CsF 80; CsCl where Y is fractional current recovery constrained to 1.0 at 40; NaCl 15; HEPES 10; EGTA 10; pH 7.35 with CsOH. infinity time, A is normalized control amplitude, X is recovery Currents were sampled at 10 kHz and filtered at 1–3 kHz ␶ time, and r is time constant of recovery, and the goodness of using an Axon 200B amplifier, digitized via a Digidata fit was compared to that of a biexponential function. The 1321A interface, and analyzed using pClamp 8.2 (Axon/ effects of anesthetics were compared to control using sum-of- Molecular Devices, Sunnyvale, CA). Capacitance and 60– squares F test between curve fits of mean data. The time 85% series resistance were compensated, and leak current course of use-dependent decay of normalized INa was ana- was subtracted using P/4 or P/5 protocols. Cells were held ϭ ϩ lyzed by fitting to the monoexponential equation INa at –80 mV between recordings. Only cells with Na cur- Ϫ␶ ϩ exp( use ·n) C, where n is pulse number, C is plateau INa, rents of 0.5–3.5 nA were analyzed to minimize increasing ␶ ϩ and use is time constant of use-dependent decay. Data were series resistance and contributions of endogenous Na analyzed using pClamp 8.2 (Axon/Molecular Devices), Prism currents (Ͻ 50 pA) occasionally observed in Chinese ham- 19 4.0 (GraphPad Software Inc., San Diego, CA), and SigmaPlot ster ovary cells. 6.0 (SPSS Science Software Inc., Chicago, IL). Curve fits were compared by sum-of-squares F test. Statistical significance was Anesthetics assessed by analysis of variance with Newman-Keuls post hoc Thymol-free halothane was obtained from Halocarbon test or paired or unpaired t tests, as appropriate; P Ͻ 0.05 was Laboratories (River Edge, NJ); isoflurane and sevoflurane considered statistically significant. were from Abbott Laboratories (Abbott Park, IL); enflurane was from Anaquest Inc. (Liberty Corner, NJ); desflurane was from Baxter Healthcare Corporation (Deer- Results field, IL). Anesthetics were diluted from saturated aqueous stock solutions made in extracellular solution Inhibition of Peak INa ϩ (14–16 mM halothane, 10–12 mM isoflurane, 4–6 mM Average peak Na current (INa) in Chinese hamster ␣ sevoflurane, 10–12 mM enflurane, 8–10 mM desflurane) ovary cells transfected with the Nav1.4 -subunit was prepared 12–24 h before experiments into airtight glass –3.0 Ϯ 0.2 nA (n ϭ 11) from a holding potential of –120 Ͻ syringes and applied locally to attached cells at 50–70 mV. Peak INa was rapidly (onset 1.5 min) and revers- ␮l/min using an ALA-VM8 pressurized perfusion system ibly inhibited by all five inhaled anesthetics tested (fig. (ALA Scientific, Westbury, NY) through a perfusion pi- 1) and by the specific Naϩ channel blocker tetrodo-

Anesthesiology, V 110, No 3, Mar 2009 584 YANG ET AL.

Fig. 2. Voltage-dependent inhibition of Nav1.4 by inhaled anes- Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/110/3/582/367953/0000542-200903000-00024.pdf by guest on 29 September 2021 thetics. Equipotent concentrations (1 minimum alveolar concentration [MAC]) of inhaled anesthetics differentially inhibited

INa 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 mM (1.0 MAC), respectively, from a holding potential of 0.03 ؎ 0.83 mV; they were 0.38 ؎ 0.05 mM (1.1 MAC), 0.41 ؎ 0.04 mM (1.1 80– MAC), 0.45 ؎ 0.05 mM (1.0 MAC), 0.80 ؎ 0.05 mM (1.1 MAC), and mM (1.0 MAC), respectively, from a holding potential of 0.04 ؎ 0.83 > P ** .(12–4 ؍ mV. Data are expressed as mean ؎ SEM (n 120– 0.01 by unpaired t test. investigated using a holding potential of –120 mV to maintain channels in the resting state, allowing assess- ment of resting channel block with minimal interference Fig. 1. Inhibition of Nav1.4 by equipotent concentrations of ؉ from voltage-dependent inactivation. All five anesthetics various inhaled anesthetics. Na currents (INa) were recorded from a holding potential of –80 mV by 25-ms test steps to Vmax exhibited concentration-dependent inhibition of peak (–10 or –20 mV) as shown in the inset. The effects of halothane INa with IC50 values in the millimolar range and Hill (A, 0.40 mM; 1.1 minimum alveolar concentration [MAC]), isoflurane (B, 0.42 mM; 1.2 MAC), and sevoflurane (C, 0.46 mM; 1.0 slopes of 2, except for halothane, which had a Hill slope MAC), enflurane (D, 0.81 mM; 1.1 MAC), and desflurane (E, 0.85 of 1 (fig. 3); this suggests the possibility of two sites of ϳ mM; 1.1 MAC) at 1 MAC are shown in these representative interaction with Na 1.4 for the ethers versus a single site traces (summary data are given in Results). The time-course of v of interaction for the alkane. INa inhibition expressed as fractional INa (INa/INa 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 was repeti- Na Effects on Channel Gating tively activated from a holding potential of –120 mV by 25-ms test pulses to –10 mV at 0.5-s intervals. None of the anesthetics tested altered the current-voltage relationship or reversal potential for I (fig. 4; data not toxin (data not shown). Inhibition was greater from Na the more physiologic 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, 20 and 0.80 mM for desflurane), desflurane showed the greatest inhibition of peak INa from a holding potential of –80 mV. The rank order of inhibition was desflurane (57 Ϯ 6.2% at 0.83 Ϯ 0.06 mM,nϭ 4) Ͼ halothane (32 Ϯ 3.5% at 0.42 Ϯ 0.05 mM,nϭ 6) Ϸ enflurane (32 Ϯ 6.7% at 0.82 Ϯ 0.06 mM,nϭ 5) Ͼ isoflurane (19 Ϯ 1.9% at 0.46 Ϯ 0.04 mM,nϭ 4) Ϸ sevoflurane (17 Ϯ 3.3% at 0.44 Ϯ 0.04 mM, n ϭ 4; mean Ϯ SEM). The degree of voltage-dependent inhibition (difference between efficacy at –80 mV vs. –120 mV) varied between anesthetics and was greatest for des- Fig. 3. Concentration dependence of Nav1.4 inhibition by in- flurane and least for halothane (fig. 2). haled anesthetics. Normalized peak INa values from a holding ϩ potential of –120 mV were fitted to the Hill equation to yield IC50 ؎ Voltage-gated Na channels have at least three distinct 13 values ( SE) and Hill slopes. IC50 values differed from each ؍ halothane; Œ ؍ ▫ .(conformational states: resting, open, and inactivated. other by sum-of-squares F test (P < 0.05 .desflurane ؍ ;enflurane ؍ ;sevoflurane ؍ The potency of each anesthetic for tonic inhibition was isoflurane; ƒ

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Fig. 4. Effects of isoflurane and halothane on Nav1.4 current- voltage relationship. (A) Isoflurane (left) or halothane (right)at concentrations equivalent to ϳ2 minimum alveolar concentration (MAC) significantly inhibited I from a holding potential Na Fig. 5. Effects of volatile anesthetics on voltage-dependence of of –120 mV. (B) Voltage of peak I activation (V ) was –10 (Na max Na 1.4 activation. Normalized conductance data (mean ؎ SEM mV; neither anesthetic affected voltage of peak current activa- v were fitted to a Boltzmann equation to yield voltage of 50% max- tion or reversal potential. Mean concentrations of isoflurane imal activation (V ) and slope factor. Holding potential was were 0.84 ؎ 0.08 mM (2.4 MAC) and of halothane was 0.81 ؎ 0.06 1/2a There were no significant shifts in V or .(12–5 ؍ mV (n 120– Comparable results were obtained for 1/2a .(8–5 ؍ mM (2.3 MAC) (n -isoflu ؍ halothane; Iso ؍ slope factor (P > 0.05 by t test). Halo desflurane, enflurane, and sevoflurane (data not shown). ***P < .desflurane ؍ enflurane; Des ؍ sevoflurane; Enf ؍ orane; Sevo 0.001, **P < 0.01 vs. control by paired t test. ϳ shown). At concentrations equivalent to 1 MAC, all five and halothane on recovery of INa from fast inactivation anesthetics produced no significant shift in the voltage were evaluated by a two-pulse protocol with varying dependence of activation (fig. 5), with minor effects on time intervals (fig. 7). Both anesthetics slowed recov- slope factors (data not shown). The voltage dependence of ery from inactivation by increasing the time constant ␶ fast inactivation was determined for the prototypical anes- of recovery ( r, ms) derived from monoexponential thetic isoflurane and for desflurane and halothane, which fits of the fractional current (fig. 7). exhibited extremes in voltage sensitivity (fig. 2). Representa- tive current traces, which reflect channel availability at various Use-dependent Block holding potentials, obtained using a protocol designed to min- Use-dependent block of Nav1.4 was evident as a reduc- imize the influence of slow inactivation are shown in figure tion in normalized INa relative to the peak of the first pulse 6A. Isoflurane, halothane, and desflurane strongly enhanced evaluated in a series of rapid depolarizing pulses (fig. 8). In inactivation in a concentration-dependent manner. Desflurane the absence of anesthetic, repetitive pulses produced only produced a greater negative shift in V1/2in than isoflurane or small reductions in peak INa. Both halothane and isoflurane halothane as seen in curve fits of the mean data (fig. 6, B and at ϳ2 MAC reduced the time constant of use-dependent C). At concentrations of ϳ1 MAC, isoflurane, halothane, and ␶ decay ( use). Halothane produced a greater reduction in desflurane shifted V1/2in by –7 mV, –9 mV, and –13 mV, Ͻ steady-state normalized INa amplitude than isoflurane (P respectively (fig. 6B). Similar effects were evident when the 0.05 by paired t test, n ϭ 3). These results are consistent data were analyzed by calculating the mean values of V1/2in with contributions of open channel block and/or enhanced derived from curve fits of the individual data sets (table 1). inactivation to inhibition of Nav1.4. Slope factors, which reflect the voltage sensitivity of the inactivation gate,21 were not significantly affected by isoflurane or halothane, but they were slightly increased by desflurane Discussion (table 1). Macroscopic current inactivation was examined by fitting the rate of decay of current elicited by depolarization We compared the actions of five potent halogenated ϩ from –120 mV to Vmax to a mono-exponential equation. Time inhaled anesthetics on a single Na channel isoform ␶ Ͼ constants of current decay ( in) were reduced by desflurane (Nav1.4) expressed in a uniform mammalian cellular envi- halothane Ͼ isoflurane (table 2). The effects of isoflurane ronment to enhance detection of possible agent–specific

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Fig. 6. Effects of isoﬂurane, halothane, and desﬂurane on voltage dependence of

Nav1.4 fast inactivation. (A) Representative traces show inhibition of INa 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 (V1/2in) and slope factor for ϳ1 minimum alveolar concentration (MAC)(B)orϳ2 MAC (C). Each anesthetic signiﬁcantly shifted the

V1/2in 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 -mM and 0.82 ؎ 0.07 mM for isoflu 0.09 ؎ orane, 0.40 ؎ 0.06 mM and 0.77 ؎ 0.10 mM for halothane, and 0.82 ؎ 0.06 mM and 1.61 -mM for desflurane. Data are ex 0.07 ؎ .7–5 ؍ pressed as mean ؎ SEM, n

effects on state-dependent inhibition. All five of these clin- a hyperpolarized holding potential of –120 mV. Members ically used inhaled anesthetics, with representatives from of the same drug class have agent-specific differences in both alkane and ether subclasses, inhibited currents con- their effects on a single target with potential pharmacolog- ␣ ϩ ducted by the -subunit of the Nav1.4 voltage-gated Na ical implications. A clinical implication of these findings is channel isoform at clinically relevant concentrations con- that inhibition of Nav1.4 could contribute to skeletal mus- sistent with a role for blockade of Nav in anesthetic immo- cle-relaxing effects of anesthetics given the high density of 1 12 bilization. There were differences between agents in their Nav1.4 at the neuromuscular junction. Indeed the greater potencies for inhibition of Nav1.4 relative to their anes- inhibition of Nav1.4 by desflurane at 1 MAC correlates with thetic potencies, and there were differences in the voltage- its relatively greater enhancement of nondepolarizing neu- dependence of their inhibition. For example, at equianes- romuscular blocking drug potency during anesthesia in thetic concentrations, desflurane was the most effective vivo in human subjects.22 inhibitor of peak INa from a near physiologic holding po- Inhibition of Nav1.4 from a hyperpolarized holding tential of –80 mV, and halothane was most effective from potential is consistent with anesthetic block of the

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Table 1. Inhaled Anesthetic Effects on Nav1.4 Inactivation nists. The relative contributions of open state block and enhanced fast and/or slow inactivation to use-dependent V ,mV k n 1/2in block by inhaled anesthetics, which differs between vari- ϳ1 MAC ous Naϩ channel blockers, should be resolvable by exam- Ϫ Ϯ Ϯ ϩ Control 49.1 1.4 6.5 0.2 4 ining anesthetic effects on fast inactivation-deficient Na Isoflurane (0.46 Ϯ 0.09 mm) Ϫ55.4 Ϯ 2.6 6.9 Ϯ 0.1 4 Control Ϫ48.8 Ϯ 2.5 7.0 Ϯ 0.5 7 channels. Halothane (0.40 Ϯ 0.06 mm) Ϫ57.8 Ϯ 2.5‡ 7.2 Ϯ 0.4 7 Inhaled anesthetics are known to inhibit various iso- Control Ϫ57.2 Ϯ 1.1 6.6 Ϯ 0.1 5 ␣ forms of Nav -subunits heterologously expressed in Desflurane (0.82 Ϯ 0.06 mm) Ϫ69.7 Ϯ 3.0†§͉͉ 7.4 Ϯ 0.3* 5 ϳ Chinese hamster ovary cells (rat Nav1.2, Nav1.4, and 2 MAC 11 Control Ϫ53.2 Ϯ 2.3 7.1 Ϯ 0.3 5 Nav1.5), human embryonic kidney cells (HEK 293, Ϯ Ϫ Ϯ Ϯ 9 Isoflurane (0.82 0.07 mm) 63.3 2.1* 8.4 0.9 5 human Nav1.5), and Xenopus oocytes (rat Nav1.2 and Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/110/3/582/367953/0000542-200903000-00024.pdf by guest on 29 September 2021 Control Ϫ54.1 Ϯ 2.2 7.3 Ϯ 0.4 5 10 1.6, human Nav1.4). Small differences in potencies Halothane (0.77 Ϯ 0.10 mm) Ϫ64.9 Ϯ 2.8‡ 7.9 Ϯ 0.6 5 Control Ϫ58.7 Ϯ 1.2 6.7 Ϯ 0.1 4 reported between various studies probably result from Desflurane (1.61 Ϯ 0.07 mm) Ϫ83.2 Ϯ 1.6‡§͉͉ 7.5 Ϯ 0.1* 4 differences in isoform sensitivity, species, expression systems, ␤-subunit coexpression, recording conditions, Data for each experiment were fitted to a standard Boltzmann equation, and stimulation protocols, anesthetic concentration determi- V and slope values for each experiment were averaged and displayed as 1/2in nations, etc. Isoflurane at clinically relevant concentra- mean Ϯ SEM. * P Ͻ 0.05, † P Ͻ0.01, ‡ P Ͻ 0.001 vs. respective control by paired t test.§ P Ͻ 0.05 vs. isoflurane, ͉͉ P Ͻ 0.05 vs. halothane by one-way analysis of tions inhibits rat neuronal (Nav1.2), skeletal muscle ϩ variance with Newman-Keuls post hoc test. k ϭ slope factor; MAC ϭ minimum (Nav1.4), and cardiac muscle (Nav1.5) voltage-gated Na alveolar concentration; V ϭ voltage of half-maximal inactivation. 1/2in channel ␣-subunits studied under identical conditions

21 with isoform-dependent differences in state-dependent closed resting state. Our results are comparable to 11 block. Significantly lower IC50 values for isoflurane were those reported for the cardiac isoform Nav1.5, for which observed at more physiologic holding potentials11 due to equianesthetic concentrations of halothane are more po- marked voltage-dependent effects on channel-gating com- tent than isoflurane in tonic blockade of human9 and 23 pared to the hyperpolarized potential used to characterize guinea pig cardiac INa. Isoflurane, halothane, and des- tonic block in the current study. The IC for inhibition of flurane, which were analyzed in more detail, exhibited 50 Nav1.4 by isoflurane reported previously for a holding po- state-dependent block and a negative shift in the voltage ϭ 11 tential of –100 mV (IC50 0.99 mM) compares well with dependence of inactivation, consistent with enhance- the value obtained in the present study for a holding po- ment of inactivation at physiologic holding potentials. ϭ tential of –120 mV (IC50 1.16 mM). Rat Nav1.8 in Xeno- State-dependent block has been reported previously for pus oocytes has been reported to be insensitive to isoflu- isoflurane effects on INa in rat neurohypophysial nerve rane,10 but recent evidence indicates that rat Na 1.8 terminals,7 guinea pig cardiomyocytes,23 and heterolo- v 8–11 expressed in a mammalian neuroblastoma cell line is inhib- gously expressed Nav1.2, Nav1.4, Nav1.5, and Nav1.6. ited by isoflurane at concentrations comparable to those Moreover, isoflurane and halothane affected channel-gat- effective on other isoforms (unpublished data, 2008; Karl F. ing, evident as accelerated current decay and use-depen- Herold, M.D., and Hugh C. Hemmings, M.D., Ph.D., New dent block (halothane Ͼ isoflurane). The similarity of these York NY). All mammalian Nav isoforms tested so far are effects of inhaled anesthetics to the effects of local anes- susceptible to inhibition by the prototypical inhaled anesthetics, antidepressants, and anticonvulsants on Nav1.2 and 13–17 thetic isoflurane with minor isoform-specific differences in Nav1.4 currents suggests conserved or overlapping relative potency and mechanism. drug binding sites and/or allosteric conformational mecha- ϩ Human Nav1.4 heterologously expressed in Xenopus nisms for these chemically diverse Na channel antago- oocytes is inhibited by isoflurane and halothane,10 whereas rat Na 1.4 in the same expression system was Table 2. Inhaled Anesthetic Effects on Na 1.4 Current Decay v v reported to be insensitive to halothane unless coex- ␶ 24 in,ms pressed with protein kinase C (PKC). Our results indi- Anesthetic cate that rat Nav1.4 expressed in a mammalian cell line is Control Anesthetic Concentration, mM n inhibited by multiple inhaled anesthetics in the absence Isoflurane 0.54 Ϯ 0.08 0.47 Ϯ 0.08* 0.46 Ϯ 0.05 8 of overexpression or pharmacological activation of PKC, 0.52 Ϯ 0.09 0.43 Ϯ 0.08* 0.85 Ϯ 0.04 6 indicating that PKC activation is apparently not required Ϯ Ϯ Ϯ Halothane 0.51 0.07 0.42 0.04* 0.43 0.03 5 for inhibition. A requirement for activation of endoge- 0.55 Ϯ 0.07 0.39 Ϯ 0.06† 0.81 Ϯ 0.04 5 Desflurane 0.55 Ϯ 0.10 0.40 Ϯ 0.05*‡ 0.80 Ϯ 0.06 7 nous PKC, which can be activated by halogenated in- 0.54 Ϯ 0.09 0.34 Ϯ 0.08†‡§ 1.65 Ϯ 0.14 5 haled anesthetics,25 cannot be excluded, however. At- tempts to test this possibility by inhibition of Values are mean Ϯ SEM. * P Ͻ 0.01, † P Ͻ 0.001 vs. respective control by endogenous PKC using small-molecule PKC inhibitors unpaired t test. ‡ P Ͻ 0.05 vs. isoflurane, § P Ͻ 0.05 vs. halothane by one-way analysis of variance with Newman-Keuls post hoc test. ␶ ϭ time constant of have been unsuccessful because the PKC inhibitors in ϩ 26 current decay calculated for a prepulse potential of –120 mV. themselves inhibit Na channels.

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Fig. 7. Effects of isoﬂurane and halothane

on recovery of Nav1.4 from fast inactivation. The protocol (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 minimum alveolar concentration [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 monoexponential function in all cases to yield a recovery time constant ␶ ( r). The rate of recovery, expressed as current normalized to initial control cur- Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/110/3/582/367953/0000542-200903000-00024.pdf by guest on 29 September 2021 rent, 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 mM and 0.82 ؎ 0.08 mM; mean 0.06 ؎ 0.44 ؎ halothane concentrations were 0.41 .P < 0.001 versus. control *** .8–5 ؍ mM and 0.78 ؎ 0.10 mM. Data are expressed as mean ؎ SEM, n 0.07

Inhaled anesthetics negatively shift the voltage depen- ical proﬁles of speciﬁc anesthetics in vivo, e.g., differen-

dence of INa fast inactivation. Similar shifts in inactiva- tial effects on brain, heart, and skeletal muscle. tion of INa are produced in ventricular cardiomyocytes All five inhaled anesthetics tested produced insignifi- 9 by isoflurane and halothane and in rat neurohypophy- cant shifts in the voltage dependence of Nav1.4 activa- 7 9 sial nerve terminals and heterologously expressed Nav tion, similar to the findings of Stadnicka et al. but in isoforms by isoflurane.10,11 Negative shifts in the voltage contrast to those of Weigt et al.,23 who found small

dependence of fast inactivation suggest greater anes- negative shifts for Nav1.5. These ﬁndings suggest iso- thetic binding afﬁnity and selective stabilization of inac- form-selective interactions between volatile anesthetics

tivated states. The large negative shift in V1/2in by des- and the activation process similar to those observed for ﬂurane contributes to its greater potency compared to the local anesthetic lidocaine.28 Another class of anes- ϩ isoﬂurane and halothane for inhibition of Nav1.4 from thetics, the n-alkanols, also inhibits voltage-gated Na the more positive (physiologic) holding potential of –80 channels at anesthetic concentrations, but with some- mV vs. –120 mV. Preferential anesthetic interaction with what distinct mechanisms from those of inhaled anes-

the nonconducting inactivated state of Nav1.4 is also thetics that involve primarily open channel block with consistent with anesthetic slowing of recovery from fast relatively small effects on both activation and inactiva- inactivation. The slightly greater slowing effect of halo- tion.29 Thus the n-alkanols and inhaled anesthetics both thane compared to isoﬂurane is consistent with a greater inhibit Naϩ channels, but they differ in the relative

shift in V1/2in and hence stronger interaction with the involvement of open channel block and activation fast inactivated state for halothane. (greater for the alkanols) versus inactivation mecha-

Enhanced inactivation is critical to inhibition of Nav1.4 nisms (greater for inhaled anesthetics). by inhaled anesthetics at more positive membrane po- Both halothane and isoflurane exhibited use-dependent tentials. This has classically been attributed to fast inac- block with repetitive stimuli. The fraction of open versus tivation, but recent evidence implicates slow inactiva- inactivated channels increases during fast repetitive depo- 4 tion in activity-dependent attenuation of Nav currents. larizations; therefore, the presence of use-dependent block The possible role of slow inactivation in the Naϩ chan- suggests a possible role for open-channel block and/or slow nel-blocking effects of inhaled anesthetics is not clear, inactivation mechanisms by both anesthetics.17,21 Halothane but it is an important area for future investigation that was more efficacious than isoflurane in inhibiting normalized

will be facilitated using mutations in Nav1.4 that enhance current amplitude with repetitive stimuli consistent with its 27 or remove inactivation. Possible mechanisms underly- greater tonic INa blocking effect. Open-channel block is par- ing state-dependent effects include facilitated transitions ticularly important in pathologic conditions such as myotonia

from the closed to inactivated state (tonic block), open and periodic paralysis, which involve Nav1.4 inactivation gat- to inactivated state (enhanced inactivation), and/or sta- ing defects,30 inﬂammatory and neuropathic pain states that 31,32 bilization of inactivated states (delayed recovery). Small involve repetitive activation of Nav1.7 and Nav1.8, and differences between anesthetics in their state-dependent ischemia, which leads to resting membrane depolarization.33 11 effects on various Nav isoforms could underlie anes- Accessory subunits can have important effects on the thetic- and isoform-speciﬁc differences in pharmacolog- pharmacological and gating properties of voltage-gated

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of MAC). We are thus unable to deﬁne the degree of Naϩ 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 Nav 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, in-

cluding the species and isoform of the channel, expres- Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/110/3/582/367953/0000542-200903000-00024.pdf by guest on 29 September 2021 sion 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 Fig. 8. Enhanced use-dependent block of Nav1.4 by isoflurane and halothane. (A) Representative current traces showing sensitivity. Given these reservations, the observation that isoflurane (left) and halothane (right; solid lines) enhancement all five inhaled anesthetics tested inhibit Nav1.4 sup- of use-dependent block of INa compared to control (dotted ports, but does not prove, an important role for Na lines) from a holding potential of –120 mV. (B) Peak currents v -were normalized to the current of the first pulse (mean ؎ SEM; inhibition in anesthesia. Additional support for this hy -plotted against pulse number, and fitted to a monoex- pothesis is provided by the recent observation that in ,(3 ؍ n ponential function. Halothane (0.82 ؎ 0.06 mM) reduced the ␶ trathecal administration of the Nav agonist veratridine time constant of use-dependent decay ( use) from 7.3 to 2.0 38 .␶ ؎ increases MAC in rats ؎ pulses, and isoflurane (0.85 0.08 mM) reduced use from 3.3 Halothane produced a In summary, halogenated inhaled anesthetics all inhibit .(3 ؍ to 1.6 ؎ 0.02 pulses (mean, n 0.02 significantly greater reduction in the plateau INa amplitude heterologously expressed Na 1.4 at clinical concentra- to 0.63 ؎ 0.02) than isoflurane (0.90 ؎ 0.01 to 0.76 v 0.02 ؎ 0.94) Repetitive pulse tions by state-dependent mechanisms. These findings .(3 ؍ P < 0.05 by paired t test, n ;0.01 ؎ protocol: 25-ms, 10-Hz test pulses from holding potential of support inhibition of Nav as a common mechanism for –120 mV to peak activation voltage. inhaled anesthetic action. Small agent-specific differences in relative potency and gating effects are consis- ϩ Na channels that must be considered in pharmacolog- tent with subtle interagent variability in pharmacody- ical studies, although ␣-subunit expression is sufficient namic profiles, such as skeletal muscle relaxant effects. ϩ 4 to mimic native Na channel-gating properties. Modu- Agent-specific differences in potency for Na 1.4 inhibi- ␤ v lation of Nav function by -subunits depends on both the tion at normal resting membrane potential were deter- ␣-subunit isoform and the cell type used for expression. mined primarily by differences in state-dependent block Coexpression of the ␤1-subunit has no effect on inhibi- reflected in effects on inactivation gating. These gating tion by isoflurane of Nav1.2, Nav1.4, Nav1.6, or Nav1.8 effects of inhaled anesthetics are remarkably similar to ␣-subunits expressed in Xenopus oocytes.10 In mamma- those of local anesthetics, and they suggest the possibil- lian expression systems, ␤1-subunit coexpression has no ity of overlapping binding sites,13–17 an interesting hy- major effects on local anesthetic sensitivity, current ki- pothesis that can now be tested by detailed structure- netics, or activation and inactivation properties of tetrodo- function studies in Nav1.4 using mutations that affect toxin-sensitive currents in ND7/23 cells,34 but it produces gating mechanisms and local anesthetic sensitivity. positive shifts of channel activation and inactivation of 35 Nav1.2 in tsA-201 cells and positive shifts of inactivation but no change in cocaine affinity of Nav1.4 and Nav1.5 in human embryonic kidney 293t cells.36 Although we have References not ruled out possible effects of ␤-subunit coexpression on 1. 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