Free
Perioperative Medicine  |   March 2009
Comparative Effects of Halogenated Inhaled Anesthetics on Voltage-gated Na+Channel Function
Author Affiliations & Notes
  • Wei Ouyang, Ph.D.
    *
  • Karl F. Herold, M.D., Ph.D.
  • Hugh C. Hemmings, M.D., Ph.D.
  • * Research Associate, ‡ Professor and Vice Chair of Research, Departments of Anesthesiology and Pharmacology, Weill Cornell Medical College, New York, New York; † Postdoctoral Associate, Department of Anesthesiology, Weill Cornell Medical College, and Department of Anesthesiology and Intensive Care, Campus Mitte, Charité University Medicine Berlin, Berlin, Germany.
Article Information
Perioperative Medicine / Pharmacology / Respiratory System
Perioperative Medicine   |   March 2009
Comparative Effects of Halogenated Inhaled Anesthetics on Voltage-gated Na+Channel Function
Anesthesiology 3 2009, Vol.110, 582-590. doi:10.1097/ALN.0b013e318197941e
Anesthesiology 3 2009, Vol.110, 582-590. doi:10.1097/ALN.0b013e318197941e
BOTH ligand-gated ion channels, including GABAA(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 (Nav) superfamily consists of 9 distinct genes that encode for the channel-forming α-subunit (Nav1.1–1.9), each with tissue-dependent expression and functions.4 The potent inhaled anesthetics inhibit native neuronal Na+channels5–7 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 Navisoforms are therefore likely to be involved in their anesthetic and some of their agent-specific nonanesthetic effects.
We characterized the effects of five potent inhaled anesthetics on the function and gating of rat Nav1.4 α-subunits heterologously expressed in Chinese hamster ovary cells. The skeletal muscle Na+channel isoform Nav1.4 is expressed at the neuromuscular junction, where it regulates muscle excitability.12 Nav1.4 function is inhibited by local anesthetic and antidepressant drugs, and it 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 for identifying common and/or distinct features that might contribute to agent-specific pharmacological profiles. We report here that all five anesthetics inhibit Nav1.4 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 Nav1.4 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) Dulbecco’s Modified Eagle 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% CO2at 37°C. Cells were plated on glass coverslips in 35-mm plastic dishes (Becton Dickinson, Franklin Lakes, NJ) 1–3 days before 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; CaCl21.5; MgCl21.5; 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 pipette 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 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); 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 before 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 perfusion pipette (diameter, 0.15 mm) 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
IC50values 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/Gmax= 1/(1 + e(V1/2a– V)/k  ), where G/Gmaxis normalized fractional conductance, Gmaxis maximum conductance, V1/2ais voltage for half-maximal activation, and k  is the slope factor. Na+conductance (GNa) was calculated using the equation: GNa=I  Na/(Vt– Vr), where I  Nais peak Na+current, Vtis test potential, and Vris Na+reversal potential (ENa= 69 mV). Fast inactivation curves were fitted to a Boltzmann equation of the form I  /I  max= 1/(1 + e(V1/2in– V)/k  ), where I  /I  maxis normalized current, I  maxis maximum current, V1/2inis voltage of half maximal inactivation, and k  is slope factor. I  Nacurrent decay was analyzed by fitting the decay phase of the current trace between 90% and 10% of maximal I  Nato the monoexponential equation I  Na= A · exp(−t/τin) + C, where A is maximal I  Naamplitude, C is plateau I  Na, t is time, and τinis time constant of current decay. Channel recovery from fast inactivation was fitted to the monoexponential function Y = A × (1 – exp(−τr× X)), where Y is fractional current recovery constrained to 1.0 at infinity time, A is normalized control amplitude, X is recovery time, and τris time constant of recovery, and the goodness of fit was compared to that of a biexponential 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  Nawas analyzed by fitting to the monoexponential equation I  Na= exp(−τuse· n) + C, where n is pulse number, C is plateau I  Na, and τuseis time constant of use-dependent decay. Data were analyzed using pClamp 8.2 (Axon/Molecular Devices), Prism 4.0 (GraphPad 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 INa
Average peak Na+current (I  Na) in Chinese hamster ovary cells transfected with the Nav1.4 α-subunit was –3.0 ± 0.2 nA (n = 11) from a holding potential of –120 mV. Peak I  Nawas 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 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, and 0.80 mm for desflurane),20 desflurane showed the greatest inhibition of peak I  Nafrom a holding potential of –80 mV. The rank order of inhibition was desflurane (57 ± 6.2% at 0.83 ± 0.06 mm, n = 4) > halothane (32 ± 3.5% at 0.42 ± 0.05 mm, n = 6) ≈ enflurane (32 ± 6.7% at 0.82 ± 0.06 mm, n = 5) > isoflurane (19 ± 1.9% at 0.46 ± 0.04 mm, n = 4) ≈ sevoflurane (17 ± 3.3% at 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 Nav1.4 by equipotent concentrations of various inhaled anesthetics. Na+currents (  INa  ) were recorded from a holding potential of –80 mV by 25-ms test steps to Vmax(–10 or –20 mV) as shown in the  inset  . The effects of halothane (  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 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 Results). The time-course of  I  Nainhibition expressed as fractional  I  Na(  I  Na  /I  Nacontrol) 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  Nawas repetitively activated from a holding potential of –120 mV by 25-ms test pulses to –10 mV at 0.5-s intervals. 
Image Not Available
Fig. 1. Inhibition of Nav1.4 by equipotent concentrations of various inhaled anesthetics. Na+currents (  INa  ) were recorded from a holding potential of –80 mV by 25-ms test steps to Vmax(–10 or –20 mV) as shown in the  inset  . The effects of halothane (  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 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 Results). The time-course of  I  Nainhibition expressed as fractional  I  Na(  I  Na  /I  Nacontrol) 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  Nawas repetitively activated from a holding potential of –120 mV by 25-ms test pulses to –10 mV at 0.5-s intervals. 
×
Fig. 2. Voltage-dependent inhibition of Nav1.4 by inhaled anesthetics. Equipotent concentrations (1 minimum alveolar concentration [  MAC  ]) of inhaled anesthetics differentially inhibited  I  Nafrom 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; they 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. 
Image Not Available
Fig. 2. Voltage-dependent inhibition of Nav1.4 by inhaled anesthetics. Equipotent concentrations (1 minimum alveolar concentration [  MAC  ]) of inhaled anesthetics differentially inhibited  I  Nafrom 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; they 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  Nawith IC50values 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 Nav1.4 for the ethers versus  a single site of interaction for the alkane.
Fig. 3. Concentration dependence of Nav1.4 inhibition by inhaled anesthetics. Normalized peak  I  Navalues from a holding potential of –120 mV were fitted to the Hill equation to yield IC50values (± SE) and Hill slopes. IC50values differed from each other by sum-of-squares F test (  P  < 0.05). □= halothane; ▴= isoflurane; ▿= sevoflurane; ♦= enflurane; ○= desflurane. 
Image Not Available
Fig. 3. Concentration dependence of Nav1.4 inhibition by inhaled anesthetics. Normalized peak  I  Navalues from a holding potential of –120 mV were fitted to the Hill equation to yield IC50values (± SE) and Hill slopes. IC50values 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 V1/2inthan isoflurane or halothane as seen in curve fits of the mean data (fig. 6, B and C). At concentrations of ∼1 MAC, isoflurane, halothane, and desflurane shifted V1/2inby –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 V1/2inderived 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 or halothane, but they 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 Vmaxto a mono-exponential equation. Time constants of current decay (τin) were reduced by desflurane > halothane > isoflurane (table 2). The effects of isoflurane and halothane on recovery of I  Nafrom 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 (τr, ms) derived from monoexponential fits of the fractional current (fig. 7).
Fig. 4. Effects of isoflurane and halothane on Nav1.4 current-voltage relationship. (  A  ) Isoflurane (  left  ) or halothane (  right  ) at concentrations equivalent to ∼2 minimum alveolar concentration (  MAC  ) significantly inhibited  I  Nafrom a holding potential of –120 mV. (  B  ) Voltage of peak  I  Naactivation (  Vmax  ) was –10 mV; neither anesthetic affected voltage of peak current activation or reversal potential. Mean concentrations of isoflurane were 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. 
Image Not Available
Fig. 4. Effects of isoflurane and halothane on Nav1.4 current-voltage relationship. (  A  ) Isoflurane (  left  ) or halothane (  right  ) at concentrations equivalent to ∼2 minimum alveolar concentration (  MAC  ) significantly inhibited  I  Nafrom a holding potential of –120 mV. (  B  ) Voltage of peak  I  Naactivation (  Vmax  ) was –10 mV; neither anesthetic affected voltage of peak current activation or reversal potential. Mean concentrations of isoflurane were 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 Nav1.4 activation. Normalized conductance data (mean ± SEM) were fitted to a Boltzmann equation to yield voltage of 50% maximal activation (  V1/2a  ) and slope factor. Holding potential was –120 mV (n = 5–12). There were no significant shifts in V1/2aor slope factor (  P  > 0.05 by  t  test).  Halo  = halothane;  Iso  = isofluorane;  Sevo  = sevoflurane;  Enf  = enflurane;  Des  = desflurane. 
Image Not Available
Fig. 5. Effects of volatile anesthetics on voltage-dependence of Nav1.4 activation. Normalized conductance data (mean ± SEM) were fitted to a Boltzmann equation to yield voltage of 50% maximal activation (  V1/2a  ) and slope factor. Holding potential was –120 mV (n = 5–12). There were no significant shifts in V1/2aor slope factor (  P  > 0.05 by  t  test).  Halo  = halothane;  Iso  = isofluorane;  Sevo  = sevoflurane;  Enf  = enflurane;  Des  = desflurane. 
×
Fig. 6. Effects of isoflurane, halothane, and desflurane on voltage dependence of Nav1.4 fast inactivation. (  A  ) Representative traces show inhibition of  I  Naby 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 ∼1 minimum alveolar concentration (  MAC  ) (B  ) or ∼2 MAC (  C  ). Each anesthetic significantly shifted the V1/2inin 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 isofluorane, 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 are expressed as mean ± SEM, n = 5–7. 
Image Not Available
Fig. 6. Effects of isoflurane, halothane, and desflurane on voltage dependence of Nav1.4 fast inactivation. (  A  ) Representative traces show inhibition of  I  Naby 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 ∼1 minimum alveolar concentration (  MAC  ) (B  ) or ∼2 MAC (  C  ). Each anesthetic significantly shifted the V1/2inin 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 isofluorane, 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 are expressed as mean ± SEM, n = 5–7. 
×
Table 1. Inhaled Anesthetic Effects on Nav1.4 Inactivation 
Image Not Available
Table 1. Inhaled Anesthetic Effects on Nav1.4 Inactivation 
×
Table 2. Inhaled Anesthetic Effects on Nav1.4 Current Decay 
Image Not Available
Table 2. Inhaled Anesthetic Effects on Nav1.4 Current Decay 
×
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 (  τr  ). 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  versus  . control. 
Image Not Available
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 (  τr  ). 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  versus  . control. 
×
Use-dependent Block
Use-dependent block of Nav1.4 was evident as a reduction in normalized I  Narelative 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 (τuse). Halothane produced a greater reduction in steady-state normalized I  Naamplitude 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 Nav1.4.
Fig. 8. Enhanced use-dependent block of Nav1.4 by isoflurane and halothane. (  A  ) Representative current traces showing isoflurane (  left  ) and halothane (  right; solid lines  ) enhancement of use-dependent block of  I  Nacompared 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 monoexponential function. Halothane (0.82 ± 0.06 mm) reduced the time constant of use-dependent decay (  τuse  ) from 7.3 to 2.0 pulses, and isoflurane (0.85 ± 0.08 mm) reduced τusefrom 3.3 ± 0.02 to 1.6 ± 0.02 pulses (mean, n = 3). Halothane produced a significantly greater reduction in the plateau  I  Naamplitude (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. 
Image Not Available
Fig. 8. Enhanced use-dependent block of Nav1.4 by isoflurane and halothane. (  A  ) Representative current traces showing isoflurane (  left  ) and halothane (  right; solid lines  ) enhancement of use-dependent block of  I  Nacompared 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 monoexponential function. Halothane (0.82 ± 0.06 mm) reduced the time constant of use-dependent decay (  τuse  ) from 7.3 to 2.0 pulses, and isoflurane (0.85 ± 0.08 mm) reduced τusefrom 3.3 ± 0.02 to 1.6 ± 0.02 pulses (mean, n = 3). Halothane produced a significantly greater reduction in the plateau  I  Naamplitude (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 (Nav1.4) 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 Nav1.4 voltage-gated Na+channel isoform at clinically relevant concentrations consistent with a role for blockade of Navin anesthetic immobilization.1 There were differences between agents in their potencies for inhibition of Nav1.4 relative to their anesthetic potencies, and there were differences in the voltage-dependence of their inhibition. For example, at equianesthetic concentrations, desflurane was the most effective inhibitor of peak I  Nafrom a near physiologic holding potential of –80 mV, and halothane was most effective from a hyperpolarized holding potential of –120 mV. 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 Nav1.4 could contribute to skeletal muscle-relaxing effects of anesthetics given the high density of Nav1.4 at the neuromuscular junction.12 Indeed the greater inhibition of Nav1.4 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 Nav1.4 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 Nav1.5, for which equianesthetic 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 physiologic holding potentials. State-dependent block has been reported previously for isoflurane effects on I  Nain rat neurohypophysial nerve terminals,7 guinea pig cardiomyocytes,23 and heterologously expressed Nav1.2, Nav1.4, Nav1.5, and Nav1.6.8–11 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 Nav1.2 and Nav1.413–17 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 Navα-subunits heterologously expressed in Chinese hamster ovary cells (rat Nav1.2, Nav1.4, and Nav1.5),11 human embryonic kidney cells (HEK 293, human Nav1.5),9 and Xenopus  oocytes (rat Nav1.2 and 1.6, human Nav1.4).10 Small differences in potencies reported between various studies probably result from differences in isoform sensitivity, species, expression systems, β-subunit coexpression, recording conditions, stimulation protocols, anesthetic concentration determinations, etc  . Isoflurane at clinically relevant concentrations inhibits rat neuronal (Nav1.2), skeletal muscle (Nav1.4), and cardiac muscle (Nav1.5) voltage-gated Na+channel α-subunits studied under identical conditions with isoform-dependent differences in state-dependent block.11 Significantly lower IC50values for isoflurane were observed at more physiologic 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 IC50for inhibition of Nav1.4 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 Nav1.8 in Xenopus  oocytes has been reported to be insensitive to isoflurane,10 but recent evidence indicates that rat Nav1.8 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). All mammalian Navisoforms tested so far are susceptible to inhibition by the prototypical inhaled anesthetic isoflurane with minor isoform-specific differences in relative potency and mechanism.
Human Nav1.4 heterologously expressed in Xenopus  oocytes is inhibited by isoflurane and halothane,10 whereas rat Nav1.4 in the same expression system was reported to be insensitive to halothane unless coexpressed with protein kinase C (PKC).24 Our results indicate that rat Nav1.4 expressed in a mammalian cell line is inhibited by multiple inhaled anesthetics in the absence of overexpression 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 because the PKC inhibitors themselves inhibit Na+channels.26 
Inhaled anesthetics negatively shift the voltage dependence of I  Nafast inactivation. Similar shifts in inactivation of I  Naare produced in ventricular cardiomyocytes by isoflurane and halothane9 and in rat neurohypophysial nerve terminals7 and heterologously expressed Navisoforms 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 V1/2inby desflurane contributes to its greater potency compared to isoflurane and halothane for inhibition of Nav1.4 from the more positive (physiologic) holding potential of –80 mV vs  . –120 mV. Preferential anesthetic interaction with the nonconducting inactivated state of Nav1.4 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 V1/2inand hence stronger interaction with the fast inactivated state for halothane.
Enhanced inactivation is critical to inhibition of Nav1.4 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 Navcurrents.4 The possible role of slow inactivation in the Na+channel-blocking effects of inhaled anesthetics is not clear, but it is an important area for future investigation that will be facilitated using mutations in Nav1.4 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 Navisoforms11 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 Nav1.4 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 Nav1.5. 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 they 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; therefore, 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  Nablocking effect. Open-channel block is particularly important in pathologic conditions such as myotonia and periodic paralysis, which involve Nav1.4 inactivation gating defects,30 inflammatory and neuropathic pain states that involve repetitive activation of Nav1.7 and Nav1.8,31,32 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 Navfunction by β-subunits depends on both the α-subunit isoform and the cell type used for expression. Coexpression of the β1-subunit has no effect on inhibition by isoflurane of Nav1.2, Nav1.4, Nav1.6, or Nav1.8 α-subunits expressed in Xenopus  oocytes.10 In mammalian expression systems, β1-subunit coexpression 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 it produces positive shifts of channel activation and inactivation of Nav1.2 in tsA-201 cells35 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 not ruled out possible effects of β-subunit coexpression on inhaled anesthetic sensitivity of Nav1.4 in Chinese hamster ovary cells, 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). We are thus 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 Navblockade (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 Nav1.4 supports, but does not prove, an important role for Navinhibition in anesthesia. Additional support for this hypothesis is provided by the recent observation that intrathecal administration of the Navagonist veratridine increases MAC in rats.38 
In summary, halogenated inhaled anesthetics all inhibit heterologously expressed Nav1.4 at clinical concentrations by state-dependent mechanisms. These findings support inhibition of Navas a common mechanism for inhaled anesthetic action. Small agent-specific differences in relative potency and gating effects are consistent with subtle interagent variability in pharmacodynamic profiles, such as skeletal muscle relaxant effects. Agent-specific differences in potency for Nav1.4 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 they suggest the possibility of overlapping binding sites,13–17 an interesting hypothesis that can now be tested by detailed structure-function studies in Nav1.4 using mutations that affect gating mechanisms and local anesthetic sensitivity.
References
Hemmings HC Jr, Akabas MH, Goldstein PA, Trudell JR, Orser BA, Harrison NL: Emerging mechanisms of general anesthetic action. Trends Pharmacol Sci 2005; 26:503–10Hemmings, HC Akabas, MH Goldstein, PA Trudell, JR Orser, BA Harrison, NL
Wu XS, Sun JY, Evers AS, Crowder M, Wu LG: Isoflurane inhibits transmitter release and the presynaptic action potential. Anesthesiology 2004; 100:663–70Wu, XS Sun, JY Evers, AS Crowder, M Wu, LG
Ouyang W, Hemmings HC Jr: Depression by isoflurane of the action potential and underlying voltage-gated ion currents in isolated rat neurohypophysial nerve terminals. J Pharmacol Exp Ther 2005; 312:801–8Ouyang, W Hemmings, HC
Goldin AL: Resurgence of sodium channel research. Annu Rev Physiol 2001; 63:871–94Goldin, AL
Ratnakumari L, Hemmings HC Jr: Inhibition of presynaptic sodium channels by halothane. Anesthesiology 1998; 88:1043–54Ratnakumari, L Hemmings, HC
Ratnakumari L, Vysotskaya TN, Duch DS, Hemmings HC Jr: Differential effects of anesthetic and nonanesthetic cyclobutanes on neuronal voltage-gated sodium channels. Anesthesiology 2000; 92:529–41Ratnakumari, L Vysotskaya, TN Duch, DS Hemmings, HC
Ouyang W, Wang G, Hemmings HC Jr: Isoflurane and propofol inhibit voltage-gated sodium channels in isolated rat neurohypophysial nerve terminals. Mol Pharmacol 2003;64: 373–81Ouyang, W Wang, G Hemmings, HC
Rehberg B, Xiao YH, Duch DS: Central nervous system sodium channels are significantly suppressed at clinical concentrations of volatile anesthetics. Anesthesiology 1996; 84:1223–33Rehberg, B Xiao, YH Duch, DS
Stadnicka A, Kwok WM, Hartmann HA, Bosnjak ZJ: Effects of halothane and isoflurane on fast and slow inactivation of human heart hH1a sodium channels. Anesthesiology 1999; 90:1671–83Stadnicka, A Kwok, WM Hartmann, HA Bosnjak, ZJ
Shiraishi M, Harris RA: Effects of alcohols and anesthetics on recombinant voltage-gated Na+ channels. J Pharmacol Exp Ther 2004; 309:987–94Shiraishi, M Harris, RA
Ouyang W, Hemmings HC Jr: Isoform-selective effects of isoflurane on voltage-gated Na+ channels. Anesthesiology 2007; 107:91–8Ouyang, W Hemmings, HC
Awad SS, Lightowlers RN, Young C, Chrzanowska-Lightowlers ZM, Lomo T, Slater CR: Sodium channel mRNAs at the neuromuscular junction: Distinct patterns of accumulation and effects of muscle activity. J Neurosci 2001; 21:8456–63Awad, SS Lightowlers, RN Young, C Chrzanowska-Lightowlers, ZM Lomo, T Slater, CR
Ragsdale DS, McPhee JC, Scheuer T, Catterall WA: Molecular determinants of state-dependent block of Na+ channels by local anesthetics. Science 1994; 265:1724–8Ragsdale, DS McPhee, JC Scheuer, T Catterall, WA
Wang GK, Quan C, Wang S: A common local anesthetic receptor for benzocaine and etidocaine in voltage-gated mu1 Na+ channels. Pflugers Arch 1998; 435:293–302Wang, GK Quan, C Wang, S
Wang SY, Nau C, Wang GK: Residues in Na+ channel D3-S6 segment modulate both batrachotoxin and local anesthetic affinities. Biophys J 2000; 79:1379–87Wang, SY Nau, C Wang, GK
Nau C, Wang SY, Wang GK: Point mutations at L1280 in Nav1.4 channel D3-S6 modulate binding affinity and stereoselectivity of bupivacaine enantiomers. Mol Pharmacol 2003; 63:1398–406Nau, C Wang, SY Wang, GK
Wang GK, Russell C, Wang SY: State-dependent block of voltage-gated Na+ channels by amitriptyline via  the local anesthetic receptor and its implication for neuropathic pain. Pain 2004; 110:166–74Wang, GK Russell, C Wang, SY
Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ: Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch 1981; 391:85–100Hamill, OP Marty, A Neher, E Sakmann, B Sigworth, FJ
West JW, Scheuer T, Maechler L, Catterall WA: Efficient expression of rat brain type IIA Na+ channel alpha subunits in a somatic cell line. Neuron 1992; 8:59–70West, JW Scheuer, T Maechler, L Catterall, WA
Taheri S, Halsey MJ, Liu J, Eger EI 2nd, Koblin DD, Laster MJ: What solvent best represents the site of action of inhaled anesthetics in humans, rats, and dogs? Anesth Analg 1991; 72:627–34Taheri, S Halsey, MJ Liu, J Eger, EI Koblin, DD Laster, MJ
Hille B: Ion Channels of Excitable Membranes, 3rd edition. Sunderland, MA, Sinauer Associates, Inc, 2001.Hille, B Sunderland, MA Sinauer Associates, Inc
Wulf H, Ledowski T, Linstedt U, Proppe D, Sitzlack D: Neuromuscular blocking effects of rocuronium during desflurane, isoflurane, and sevoflurane anaesthesia. Can J Anaesth 1998; 45:526–32Wulf, H Ledowski, T Linstedt, U Proppe, D Sitzlack, D
Weigt HU, Kwok WM, Rehmert GC, Turner LA, Bosnjak ZJ: Voltage-dependent effects of volatile anesthetics on cardiac sodium current. Anesth Analg 1997; 84:285–93Weigt, HU Kwok, WM Rehmert, GC Turner, LA Bosnjak, ZJ
Mounsey JP, Patel MK, Mistry D, John JE, Moorman JR: Protein kinase C co-expression and the effects of halothane on rat skeletal muscle sodium channels. Br J Pharmacol 1999; 128:989–98Mounsey, JP Patel, MK Mistry, D John, JE Moorman, JR
Hemmings HC Jr, Adamo AIB: Activation of endogenous protein kinase C by halothane in synaptosomes. Anesthesiology 1996; 84:652–62Hemmings, HC Adamo, AIB
Ratnakumari L, Vysotskaya TN, Duch DS, Hemmings HC: Inhibition of voltage-dependent sodium channels by Ro 31-8220, a ‘specific’ protein kinase C inhibitor. FEBS Lett 2000;473: 265–8Ratnakumari, L Vysotskaya, TN Duch, DS Hemmings, HC
O’Reilly JP, Wang SY, Wang GK: Residue-specific effects on slow inactivation at V787 in D2-S6 of Na (v) 1.4 sodium channels. Biophys J 2001; 81:2100–11O’Reilly, JP Wang, SY Wang, GK
Hofer D, Lohberger B, Steinecker B, Schmidt K, Quasthoff S, Schreibmayer W: A comparative study of the action of tolperisone on seven different voltage dependent sodium channel isoforms. Eur J Pharmacol 2006; 538:5–14Hofer, D Lohberger, B Steinecker, B Schmidt, K Quasthoff, S Schreibmayer, W
Horishita T, Harris RA: n-Alcohols inhibit voltage-gated Na+ channels expressed in Xenopus  oocytes. J Pharmacol Exp Ther 2008; 326:270–7Horishita, T Harris, RA
Cannon SC: Pathomechanisms in channelopathies of skeletal muscle and brain. Annu Rev Neurosci 2006; 29:387–41Cannon, SC
Benarroch EE: Sodium channels and pain. Neurology 2007; 68:233–6Benarroch, EE
Jarvis MF, Honore P, Shieh CC, Chapman M, Joshi S, Zhang XF, Kort M, Carroll W, Marron B, Atkinson R, Thomas J, Liu D, Krambis M, Liu Y, McGaraughty S, Chu K, Roeloffs R, Zhong C, Mikusa JP, Hernandez G, Gauvin D, Wade C, Zhu C, Pai M, Scanio M, Shi L, Drizin I, Gregg R, Matulenko M, Hakeem A, Gross M, Johnson M, Marsh K, Wagoner PK, Sullivan JP, Faltynek CR, Krafte DS: A-803467, a potent and selective Nav1.8 sodium channel blocker, attenuates neuropathic and inflammatory pain in the rat. Proc Natl Acad Sci U S A 2007; 104:8520–5Jarvis, MF Honore, P Shieh, CC Chapman, M Joshi, S Zhang, XF Kort, M Carroll, W Marron, B Atkinson, R Thomas, J Liu, D Krambis, M Liu, Y McGaraughty, S Chu, K Roeloffs, R Zhong, C Mikusa, JP Hernandez, G Gauvin, D Wade, C Zhu, C Pai, M Scanio, M Shi, L Drizin, I Gregg, R Matulenko, M Hakeem, A Gross, M Johnson, M Marsh, K Wagoner, PK Sullivan, JP Faltynek, CR Krafte, DS
Hemmings HC Jr: Neuroprotection by Na+ channel blockade. J Neurosurg Anesthesiol 2004;16: 100–1Hemmings, HC
Leffler A, Reiprich A, Mohapatra DP, Nau C: Use-dependent block by lidocaine but not amitriptyline is more pronounced in tetrodotoxin (TTX)-resistant Nav1.8 than in TTX-sensitive Na+ channels. J Pharmacol Exp Ther 2007; 320:354–64Leffler, A Reiprich, A Mohapatra, DP Nau, C
Qu Y, Curtis R, Lawson D, Gilbride K, Ge P, DiStefano PS, Silos-Santiago I, Catterall WA, Scheuer T: Differential modulation of sodium channel gating and persistent sodium currents by the beta1, beta2, and beta3 subunits. Mol Cell Neurosci 2001; 18:570–80Qu, Y Curtis, R Lawson, D Gilbride, K Ge, P DiStefano, PS Silos-Santiago, I Catterall, WA Scheuer, T
Wright SN, Wang SY, Xiao YF, Wang GK: State-dependent cocaine block of sodium channel isoforms, chimeras, and channels coexpressed with the beta1 subunit. Biophys J 1999; 76:233–45Wright, SN Wang, SY Xiao, YF Wang, GK
Scholz A, Kuboyama N, Hempelmann G, Vogel W: Complex blockade of TTX-resistant Na+ currents by lidocaine and bupivacaine reduce firing frequency in DRG neurons. J Neurophysiol 1998; 79:1746–54Scholz, A Kuboyama, N Hempelmann, G Vogel, W
Zhang Y, Sharma M, Eger EI 2nd, Laster MJ, Hemmings HC Jr, Harris RA: Intrathecal veratridine administration increases minimum alveolar concentration in rats. Anesth Analg 2008; 107:875–8Zhang, Y Sharma, M Eger, EI Laster, MJ Hemmings, HC Harris, RA
Fig. 1. Inhibition of Nav1.4 by equipotent concentrations of various inhaled anesthetics. Na+currents (  INa  ) were recorded from a holding potential of –80 mV by 25-ms test steps to Vmax(–10 or –20 mV) as shown in the  inset  . The effects of halothane (  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 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 Results). The time-course of  I  Nainhibition expressed as fractional  I  Na(  I  Na  /I  Nacontrol) 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  Nawas repetitively activated from a holding potential of –120 mV by 25-ms test pulses to –10 mV at 0.5-s intervals. 
Image Not Available
Fig. 1. Inhibition of Nav1.4 by equipotent concentrations of various inhaled anesthetics. Na+currents (  INa  ) were recorded from a holding potential of –80 mV by 25-ms test steps to Vmax(–10 or –20 mV) as shown in the  inset  . The effects of halothane (  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 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 Results). The time-course of  I  Nainhibition expressed as fractional  I  Na(  I  Na  /I  Nacontrol) 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  Nawas repetitively activated from a holding potential of –120 mV by 25-ms test pulses to –10 mV at 0.5-s intervals. 
×
Fig. 2. Voltage-dependent inhibition of Nav1.4 by inhaled anesthetics. Equipotent concentrations (1 minimum alveolar concentration [  MAC  ]) of inhaled anesthetics differentially inhibited  I  Nafrom 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; they 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. 
Image Not Available
Fig. 2. Voltage-dependent inhibition of Nav1.4 by inhaled anesthetics. Equipotent concentrations (1 minimum alveolar concentration [  MAC  ]) of inhaled anesthetics differentially inhibited  I  Nafrom 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; they 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. 
×
Fig. 3. Concentration dependence of Nav1.4 inhibition by inhaled anesthetics. Normalized peak  I  Navalues from a holding potential of –120 mV were fitted to the Hill equation to yield IC50values (± SE) and Hill slopes. IC50values differed from each other by sum-of-squares F test (  P  < 0.05). □= halothane; ▴= isoflurane; ▿= sevoflurane; ♦= enflurane; ○= desflurane. 
Image Not Available
Fig. 3. Concentration dependence of Nav1.4 inhibition by inhaled anesthetics. Normalized peak  I  Navalues from a holding potential of –120 mV were fitted to the Hill equation to yield IC50values (± SE) and Hill slopes. IC50values differed from each other by sum-of-squares F test (  P  < 0.05). □= halothane; ▴= isoflurane; ▿= sevoflurane; ♦= enflurane; ○= desflurane. 
×
Fig. 4. Effects of isoflurane and halothane on Nav1.4 current-voltage relationship. (  A  ) Isoflurane (  left  ) or halothane (  right  ) at concentrations equivalent to ∼2 minimum alveolar concentration (  MAC  ) significantly inhibited  I  Nafrom a holding potential of –120 mV. (  B  ) Voltage of peak  I  Naactivation (  Vmax  ) was –10 mV; neither anesthetic affected voltage of peak current activation or reversal potential. Mean concentrations of isoflurane were 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. 
Image Not Available
Fig. 4. Effects of isoflurane and halothane on Nav1.4 current-voltage relationship. (  A  ) Isoflurane (  left  ) or halothane (  right  ) at concentrations equivalent to ∼2 minimum alveolar concentration (  MAC  ) significantly inhibited  I  Nafrom a holding potential of –120 mV. (  B  ) Voltage of peak  I  Naactivation (  Vmax  ) was –10 mV; neither anesthetic affected voltage of peak current activation or reversal potential. Mean concentrations of isoflurane were 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 Nav1.4 activation. Normalized conductance data (mean ± SEM) were fitted to a Boltzmann equation to yield voltage of 50% maximal activation (  V1/2a  ) and slope factor. Holding potential was –120 mV (n = 5–12). There were no significant shifts in V1/2aor slope factor (  P  > 0.05 by  t  test).  Halo  = halothane;  Iso  = isofluorane;  Sevo  = sevoflurane;  Enf  = enflurane;  Des  = desflurane. 
Image Not Available
Fig. 5. Effects of volatile anesthetics on voltage-dependence of Nav1.4 activation. Normalized conductance data (mean ± SEM) were fitted to a Boltzmann equation to yield voltage of 50% maximal activation (  V1/2a  ) and slope factor. Holding potential was –120 mV (n = 5–12). There were no significant shifts in V1/2aor slope factor (  P  > 0.05 by  t  test).  Halo  = halothane;  Iso  = isofluorane;  Sevo  = sevoflurane;  Enf  = enflurane;  Des  = desflurane. 
×
Fig. 6. Effects of isoflurane, halothane, and desflurane on voltage dependence of Nav1.4 fast inactivation. (  A  ) Representative traces show inhibition of  I  Naby 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 ∼1 minimum alveolar concentration (  MAC  ) (B  ) or ∼2 MAC (  C  ). Each anesthetic significantly shifted the V1/2inin 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 isofluorane, 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 are expressed as mean ± SEM, n = 5–7. 
Image Not Available
Fig. 6. Effects of isoflurane, halothane, and desflurane on voltage dependence of Nav1.4 fast inactivation. (  A  ) Representative traces show inhibition of  I  Naby 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 ∼1 minimum alveolar concentration (  MAC  ) (B  ) or ∼2 MAC (  C  ). Each anesthetic significantly shifted the V1/2inin 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 isofluorane, 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 are expressed as mean ± SEM, n = 5–7. 
×
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 (  τr  ). 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  versus  . control. 
Image Not Available
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 (  τr  ). 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  versus  . control. 
×
Fig. 8. Enhanced use-dependent block of Nav1.4 by isoflurane and halothane. (  A  ) Representative current traces showing isoflurane (  left  ) and halothane (  right; solid lines  ) enhancement of use-dependent block of  I  Nacompared 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 monoexponential function. Halothane (0.82 ± 0.06 mm) reduced the time constant of use-dependent decay (  τuse  ) from 7.3 to 2.0 pulses, and isoflurane (0.85 ± 0.08 mm) reduced τusefrom 3.3 ± 0.02 to 1.6 ± 0.02 pulses (mean, n = 3). Halothane produced a significantly greater reduction in the plateau  I  Naamplitude (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. 
Image Not Available
Fig. 8. Enhanced use-dependent block of Nav1.4 by isoflurane and halothane. (  A  ) Representative current traces showing isoflurane (  left  ) and halothane (  right; solid lines  ) enhancement of use-dependent block of  I  Nacompared 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 monoexponential function. Halothane (0.82 ± 0.06 mm) reduced the time constant of use-dependent decay (  τuse  ) from 7.3 to 2.0 pulses, and isoflurane (0.85 ± 0.08 mm) reduced τusefrom 3.3 ± 0.02 to 1.6 ± 0.02 pulses (mean, n = 3). Halothane produced a significantly greater reduction in the plateau  I  Naamplitude (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. 
×
Table 1. Inhaled Anesthetic Effects on Nav1.4 Inactivation 
Image Not Available
Table 1. Inhaled Anesthetic Effects on Nav1.4 Inactivation 
×
Table 2. Inhaled Anesthetic Effects on Nav1.4 Current Decay 
Image Not Available
Table 2. Inhaled Anesthetic Effects on Nav1.4 Current Decay 
×