Voltage-gated sodium channels have an important role in the action potential initiation and propagation in excitable cells of nerve and muscle. They are established targets of local anesthetics, but have been considered to be resistant to general anesthetics.1 However, recent reports suggest that clinical concentrations of volatile anesthetics significantly suppress sodium channel function in neurons,2,3 and in recombinant mammalian sodium channels.4–6 Moreover, blocking of presynaptic sodium channels provides a mechanism for suppression of neurotransmitter release by volatile anesthetics.7–10
N-methyl-d-aspartate (NMDA) and γ-aminobutyric acid A (GABAA) receptors are targets for general anesthetics.11–14 Although not used as clinical anesthetics, volatile aromatic compounds have industrial applications, are drugs of abuse, and have anesthetic effects.15 They inhibit NMDA receptor function,16 and enhance GABAA receptor function,17 doing so in a reciprocal manner: the aromatic anesthetics which inhibit NMDA receptors best enhance GABAA receptor function the least. The importance of aromatic structure to an effect on sodium channels is unknown and such knowledge might be important to an understanding of potential targets and mechanisms of volatile anesthetics.
Nine distinct pore-forming α subunits of sodium channels have been identified.18,19 Each α subunit has a different pattern of development and localization and demonstrates subtle differences in electrophysiological characteristics.20 We examined the effect of several structurally distinct volatile aromatics on Nav1.2, a sodium channel expressed primarily in the central nervous system, including the spinal cord, a major site of inhaled anesthetic action.21 We used the oocyte expression system to explore the mechanism of effect by volatile anesthetics on Nav1.2.
The Animal Care and Use Committees of the University of Texas approved this study.
Adult female Xenopus laevis frogs were obtained from Xenopus Express, Inc. (Plant City, FL). All aromatic anesthetics were purchased from Sigma-Aldrich (St. Louis, MO). We thank the following researchers for providing the subunit clones: cDNA for rat Nav1.2 α subunit (Dr. W. A. Catterall, University of WA, Seatle, WA), cDNA for human β1 subunit (Dr. AL George, Vanderbilt University).
Linearized cDNA templates with ClaI (Nav1.2 α subunit), EcoRI (β1 subunit) were transcribed using T7 (Nav1.2 α subunit), T6 (β1 subunit) RNA polymerase of mMESSAGE mMACHINE kit (Ambion, Ausin, TX). Preparation of Xenopus laevis oocytes and microinjection of the cRNA were performed as described previously.5 Stage IV-VI oocytes were manually isolated from an ovary. Oocytes were treated with collagenase (0.5 mg/mL) for 10 min and Nav1.2 α subunit cRNA was co-injected with the β1 subunit cRNA at a ratio of 1:10 (total volume was 20 approximately 40 ng/50 nL). Injected oocytes were incubated in modified Barth’s solution (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 10 mM HEPES, 0.82 mM MgSO4, 0.33 mM Ca(NO3)2, 0.91 mM CaCl2, adjusted to pH 7.5) supplemented with 10,000 U of penicillin, 50 mg of gentamicin, 90 mg of theophylline, and 220 mg of sodium pyruvate per liter (incubation medium) at 19°C.
Two to 6 days after injection, electrophysiological recordings were performed at room temperature (23°C) using the whole-cell, two-electrode, voltage-clamp technique. Oocytes were placed in a 100 μL recording chamber and impaled with recording electrodes prepared with borosilcate glass using a puller (P-97; Sutter Instruments Company, Novato, CA) and filled with 3M KCl/0.5% low melting point agarose and with resistances between 0.3 and 0.5 M[Omega]. The oocytes were perfused at 2 mL/min with frog Ringer’s solution containing NaCl 115 mM, KCl 2.5 mM, HEPES 10 mM, CaCl2 1.8 mM, pH 7.2 using a peristaltic pump (Cole-Palmer Instrument CO, Chicago, IL). The whole-cell voltage clamp was achieved through these two electrodes using a Warner Instruments model OC-725C (Hamden, CT). Currents were recorded and analyzed using pCLAMP software (Axon Instruments, Foster City, CA). The sodium current amplitudes were typically 2 to 15 μA. Transients and leak currents were subtracted using the P/N procedure.
The voltage dependence of activation was determined by 50-ms depolarizing pulses from a holding potential of −90 mV (VH-90) or from a holding potential causing half-maximal current (V1/2) (approximately from −50 mV to −60 mV) to 60 mV in 10-mV increments. V1/2 value was determined individually for each oocyte in each experiment. Normalized activation data were fitted to a Boltzmann equation: G/Gmax = 1/(1 + exp(V1/2 – V)/k), where G is the voltage-dependent sodium conductance, Gmax is the maximal sodium conductance, G/Gmax is the normalized fractional conductance, V1/2 is the potential at which activation is half maximal, k is the slope factor. G values for each oocyte were calculated using: G = I/(Vt – Vr), where I is the peak sodium current, Vt is the test potential, and Vr is the reversal potential. Vr for each oocyte was estimated by extrapolating the linear ascending segment of the current voltage relationship (I–V) curve to the voltage axis. To measure steady-state inactivation, currents were elicited by a 50-ms test pulse to −20 mV after 200-ms prepulses ranging from −140 mV to 0 mV in 10-mV increments from a holding potential of −90 mV. Steady-state inactivation curves were fitted to a Boltzmann equation: I/Imax = 1/(1 + exp(V1/2 – V)/k), where Imax is the maximal sodium current, I/Imax is the normalized current, V1/2 is the voltage of half-maximal inactivation, and k is the slope factor.
The aromatic anesthetics were examined at the concentrations that are equivalent to 1 MAC (minimum alveolar anesthetic concentration) or 2 MAC in rats.16,22 (MAC is the minimum anesthetic concentration needed to produce immobility in response to noxious stimulation in 50% of rats; it is equivalent to the EC50 for producing surgical immobility). All aromatic anesthetics were directly dissolved in the frog Ringer’s solution, and were infused for 3 min to produce equilibrium. The loss of concentration from vial to bath was approximately 60% to 80% for all aromatic anesthetics, as determined by gas chromatography. Bath/vial concentrations of the four compounds that were tested were 1.1/2.5 mM (benzene), 0.40/1.4 mM (1,3,5-trifluorobenzene), 0.65/2.1 mM (pentafluorobenzene), and 0.12/0.58 mM (hexafluorobenzene).
All values are presented as the mean ± sem. The n values refer to the number of oocytes studied. Each experiment was performed with oocytes from at least two frogs. Statistical analyses were performed using a one-way analysis of variance for multiple comparisons and a t-test using GraphPad Prism software (GraphPad Software Inc., San Diego, CA).
Effects of Aromatic Anesthetics on the Peak Na+ Inward Currents Elicited from Two Different Holding Potentials
All eight aromatic anesthetics suppressed INa (sodium currents) at the V1/2 holding potential, and the magnitudes of these effects varied widely (Figs. 1B and D and Fig. 2). At V1/2, these anesthetics reduced INa by 37% ± 2% (benzene), 39% ± 2% (fluorobenzene), 48% ± 2% (1,2-difluorobenzene), 31% ± 3% (1,4-difluorobenzene), 33% ± 1% (1,2,4-trifluorobenzene), 8% ± 2% (1,3,5-trifluorobenzene), 13% ± 2% (pentafluorobenzene), and 13% ± 2% (hexafluorobenzene) (Fig. 2). Effects on INa at VH-90 were smaller than at V1/2holding potential, and the magnitudes of the effects varied widely, with inhibitions of 8% ± 1% (benzene), 10% ± 1% (fluorobenzene), 15% ± 2% (1,2-difluorobenzene), 6% ± 0.5% (1,4-difluorobenzene), and 3% ± 0.7% (1,2,4-trifluorobenzene) (Fig. 1A, Fig. 2). 1,3,5-trifluorobenzene did not affect these currents. In contrast, pentafluorobenzene and hexafluorobenzene enhanced INa at VH-90 by 2% ± 1%, 6% ± 1%, respectively (Fig. 1C, Fig. 2). Thus, there are two effects of aromatic anesthetics on sodium channels: all produced inhibitory effects at V1/2 holding potential, albeit by widely varying amounts. At VH-90, benzene, fluorobenzene, 1,2-difluorobenzene, 1,4-difluorobenzene, and 1,2, 4-trifluorobenzene produced inhibition but pentafluorobenzene and hexafluorobenzene caused slight enhancement.
Effects of Aromatic Anesthetics on Activation of Sodium Currents
Because of the different effects of aromatic anesthetics noted above, we examined the actions of these two types of compounds on gating of sodium channels. We selected benzene and 1,2-difluorobenzene as compounds with potent inhibitory effects at V1/2 holding potential plus smaller inhibitory effects at VH-90, and hexafluorobenzene as the compound with a much smaller inhibitory effect at V1/2 holding potential and slight enhancing effect at VH-90. The voltage dependence of activation was determined by 50-ms depolarizing pulses from a holding potential of −90 mV to 60 mV in 10-mV increments or from a holding potential of V1/2 to 60 mV in 10-mV increments. The activation curves were derived from the I–V curve (see Methods). Benzene and 1,2-difluorobenzene (2 MAC) reduced the peak INa at VH-90 and V1/2 (Figs. 3A and B). However, hexafluorobenzene (2 MAC) enhanced the peak INa at VH-90, especially in the depolarizing region where channel opening begins (around −40 approximately −30 mV) and suppressed the sodium currents at V1/2 more weakly than benzene or 1,2-difluorobenzene (Fig. 3C). Benzene and 1,2-difluorobenzene did not affect voltage dependence of activation at VH-90 or V1/2 (Figs. 4A and B, Table 1), but hexafluorobenzene shifted the midpoint of steady-state activation, V1/2 in a hyperpolarizing direction by 4.9 and 2.2 mV at VH-90 and V1/2 (Fig. 4C, Table 1).
Effects of Aromatic Anesthetics on Inactivation of Sodium Currents
The effects of benzene, 1,2-difluorobenzene and hexafluorobenzene on steady-state inactivation were also investigated. Benzene and 1,2-difluorobenzene shifted V1/2 in the hyperpolarizing direction significantly by 7.4 and 8.9 mV, respectively and also reduced sodium currents in the hyperpolarizing potential range (Fig. 5, Table 1). Hexafluorobenzene also significantly shifted V1/2 in the hyperpolarizing direction by 4.6 mV although the shift was smaller than that by benzene and 1,2-difluorobenzene, whereas hexafluorobenzene enhanced sodium current in the hyperpolarizing potential range slightly (Fig. 5, Table 1). The effects of benzene, 1,2-difluorobenzene and hexafluorobenzene in the hyperpolarizing range were consistent with the effects of these compounds on INa at VH-90, and the effects on these compounds on activation curve at VH-90.
We found that structurally distinct aromatic anesthetics differently affected INa at VH-90 and V1/2 holding potentials. At V1/2 holding potential, 1 MAC benzene, fluorobenzene, 1,2-difluorobenzene, 1,4-difluorobenzene, 1,2,4-trifluorobenzene suppressed INa by 30% to 50%. These inhibitory effects parallel those of other inhaled anesthetics. For example, clinically relevant concentrations (0.25–0.4 mM) of isoflurane suppress INa by 30% to 40% in several preparations: Chinese hamster ovary cells expressing Nav1.2,4 isolated rat neurohypophysial nerve terminals,3 and an oocyte system expressing Nav1.2, Nav1.4, or Nav1.6.5
Solt et al. separated these aromatic anesthetics into two groups based on the inhibition of NMDA receptors produced by 1 MAC.16 Fewer fluorinated compounds with the limited fluorination, benzene, fluorobenzene, and 1,2-difluorobenzene, inhibited strongly (Group 1), and compounds with greater fluorination, 1,4-difluorobenzene, 1,2,4-trifluorobenzene, 1,3,5-trifluorobenzene, pentafluorobenzene, hexafluorobenzene, inhibited weakly (Group 2). Solt et al. also studied the effect of these compounds on GABAA receptors,17 and found that aromatic anesthetics which inhibit NMDA receptor currents most (Group 1) enhance GABAA receptor currents least, and anesthetics that inhibit NMDA receptor currents least (Group 2) enhance GABAA receptor currents most. These findings suggest that distinct molecular properties determine the action of anesthetics on NMDA and GABAA receptors.
We found that benzene, fluorobenzene, and 1,2-difluorobenzene (Group 1 compounds), strongly inhibited sodium currents at V1/2, whereas 1,3,5-trifluorobenzene, pentafluorobenezene, and hexafluorobenzene (Group 2 compounds), inhibited weakly. But we found that, unlike the NMDA results, 1,4-difluorobenzene and 1,2,4-trifluorobenzene (Group 2 compounds) inhibited sodium currents (Fig. 6A). Our previous study of isoflurane5 found similar sodium channel inhibition as for 1,4-difluorobenzene and 1,2,4-trifluorobenzene, and these three compounds are effective on both sodium channels and GABAA receptors (Fig. 6B).
Raines et al. showed that cation-π interactions predict the actions of aromatic anesthetics on NMDA receptors.23 In order to explore the physical properties important for actions of these compounds on sodium channels, we correlated drug effects with several variables (Figs. 6C–E). Sodium channel inhibition was correlated with cation-π interactions, but this relationship was weaker than seen for cation-π interactions and NMDA receptor inhibition by Raines et al.23 Inhibition of sodium channels was strongly correlated with oil/gas partition coefficients. These results suggest that aromatic anesthetics interact with different channels by distinct mechanisms. We recently reported that n-alcohols inhibit sodium channel function and their potency increase with carbon number up to a cut-off at nonanol,24 suggesting that hydrophobic and steric interactions determine the potencies of n-alcohols. Hydrophobic and steric interactions might also be important for sodium channel inhibition by aromatic anesthetics, with a smaller contribution from cation-π interactions. Some fluorinated compounds are either not as potent anesthetics as would be predicted from their lipid solubility or do not produce anesthesia (nonimmobilizers).25 The nonimmobilizing fluorinated cyclobutanes do not inhibit sodium channels,26 indicating that factors other than lipid solubility are also important for channel inhibition by some compounds.
Analysis of gating variables revealed differences in the actions of benzene, 1,2-fluorobenzene, and hexafluorobenzene. Other volatile anesthetics generally enhance inactivation with no effect on activation.5,6 We also found this to be true for benzene and 1,2-difluorobenzene. However, hexafluorobenzene enhanced peak INa at VH-90, shifted activation in the hyperpolarizing direction and increased sodium currents in the hyperpolarizing range of inactivation. These changes indicate that hexafluorobenzene shifts channel gating equilibrium toward the open channel state. No other anesthetics have been reported to have an activating effect on sodium channels. This action of hexafluorobenzene on channel gating might offset the effects on the inactivated state and explain the small inhibition at V1/2 holding potential relative to the other compounds. Pentafluorobenzene, another Group 2 compound, may shift GABAA receptor gating toward the open channel state because pentafluorobenzene decreases the GABA EC50 and activates GABAA receptors directly.17 This observation suggests some commonalities in the action of aromatic compounds on sodium channels and GABAA receptors.
In conclusion, we find that volatile aromatic anesthetics inhibit Nav1.2 sodium channel function but that the magnitude of the inhibition varies widely among the compounds. Differences in the actions of these compounds on sodium channel gating are likely responsible for the range of inhibitory effects. Although numerous inhaled anesthetics and n-alcohols inhibit sodium channel function, some inhibit weakly and others strongly (Group 1 versus Group 2) at concentrations corresponding to MAC. We applied aromatic compounds in the present study because they reciprocally affect GABAA and NMDA receptors; we tested whether the sodium channel might provide more consistent responses. However, neither the aromatics nor clinical anesthetics (e.g., halothane, isoflurane) provide consistent inhibition of sodium channels. This indicates that sodium channel inhibition may be important for some anesthetics but not others as a mediator of the capacity of inhaled anesthetics to produce immobility in the face of noxious stimulation.
We thank Dr. Michael Laster for determining the concentrations of anesthetics used in this study.
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