Isoflurane Inhibits the Tetrodotoxin-resistant Voltage-gated Sodium Channel Na^v1.8

Rat Na^v1.8-cDNA was subcloned into the mammalian expression vector pCMV-Script (Stratagene, La Jolla, CA) and the sequence of the entire Na^v1.8 channel protein (NCBI nucleotide access number U53833) was verified by dideoxynucleotide sequencing. The ND7/23 rat DRG/mouse neuroblastoma fusion cell line was purchased from Sigma (Sigma-Aldrich, St. Louis, MO) and cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Invitrogen), 2 mm l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen) at 37°C under 95% air/5% CO². Cells were grown on 12-mm glass coverslips in 35-mm polystyrene culture dishes and transiently transfected with rat Na^v1.8-pCMV-Script (1–3 μg) and the pEGFP-N1 (Clontech, Mountain View, CA) reporter plasmid (0.5–1 μg) or reporter plasmid alone using Lipofectamine LTX (Invitrogen). At 48 h after transfection, the transfected cells were identified by expression of enhanced green fluorescent protein (EGFP) by using fluorescence microscopy. Average transfection efficiency was 60–70% for Na^v1.8/EGFP. Untransfected ND7/23 cells were plated and seeded as described above and incubated for 48 h to study endogenous TTX-s Na⁺channels.

Coverslips containing ND7/23 cells were transferred into a small-volume open bath perfusion chamber (Warner Instruments, Hamden, CT) and continuously perfused with external solution containing (in mm): 129 NaCl, 10 HEPES, 3.25 KCl, 2 MgCl², 2 CaCl², 20 tetraethylammonium-Cl, 5 d-glucose, 0.0003 tetrodotoxin (Sankyo Kasei Co., Tokyo, Japan), adjusted to pH 7.4 (with NaOH) and 310 mOsm/kg H²O. Voltage-clamp recordings were performed at room temperature (23–24°C) in standard whole-cell configuration32using an Axopatch 200B patch-clamp amplifier (Molecular Devices, Sunnyvale, CA). Experiments were performed at room temperature to minimize anesthetic losses, maximize recording stability, and facilitate comparisons to other studies of isoflurane on Na⁺currents. Fire-polished patch pipettes were pulled from borosilicate glass capillaries (Warner Instruments) by using a Sutter P-97 puller (Sutter Instruments, Novato, CA) and had a resistance of 1.0–2.0 MΩ when filled with the following internal pipette solution (in mm): 120 CsF, 10 NaCl, 10 HEPES, 11 EGTA, 10 tetraethylammonium-Cl, 1 CaCl², 1 MgCl², adjusted to pH 7.3 (with CsOH) and 315 mOsm/kg H²O.

Currents were low-pass filtered at 5 kHz and sampled at 20 kHz. Capacitive transients were electronically cancelled, and voltage errors were minimized by using 70–80% series resistance compensation. Series resistance was typically 1–5 MΩ, and recordings were discarded if resistance exceeded 8 MΩ. Initial seal after establishing the whole-cell patch was 2–4 GΩ, and recordings were discarded if the seal dropped below 1 GΩ. Liquid junction potential was not corrected. Linear leakage currents were digitally subtracted online by the P/4 protocol (except for inactivation and use-dependent experiments).

Isoflurane-saturated external solutions (containing 12–12.5 mm isoflurane) were prepared by shaking in gas-tight glass vials for 24 h as previously described.16This stock solution was further diluted on the day of the experiment into gas-tight glass syringes, from which a sample was taken for determination of aqueous isoflurane concentration by gas chromatography. Solutions were perfused focally onto recorded cells via  a 150-μm diameter perfusion pipette by using polytetrafluoroethylene tubing to minimize isoflurane loss. Perfusate samples were also taken to determine isoflurane concentrations at the tip of the perfusion manifold, and reflected approximately 10% loss that occurred from the syringe through the tubing to the pipette tip. Isoflurane concentrations were determined by extraction into n -heptane (1:1 v/v) followed by analysis using a Shimadzu GC-8A gas chromatograph (Shimadzu, Tokyo, Japan) with external standard calibration as described.16,20Isoflurane solution flow of 0.05 ml/min was controlled by a pressurized perfusion system (ALA Scientific, Westbury, NY).

The holding potential (V^h) used was either −70 mV or −140 mV. To analyze the voltage-dependence of activation, currents were evoked by 5-ms pulses ranging from −80 to +70 mV in steps of 10 mV. The conductance (G  ^Na) was calculated by using the equation: G  ^Na=I  ^Na/ (V^m− V^rev), where I  ^Nais peak current, V^mis the test potential, and V^revis the calculated reversal potential (+65 mV). Normalized conductance (G /G  ^max) was plotted against test potentials and fitted to the Boltzmann function: G /G  ^max= 1/[1 + exp(V^1/2− V/k )], where V^1/2is the voltage that elicits half-maximal activation and k  is the slope factor. Steady-state and fast inactivation were measured by applying a double-pulse protocol that consisted of a 500 ms (h  ^∞) or 15 ms (fast inactivation) prepulse ranging from −120 to + 20 mV in steps of 10 mV, followed by a test pulse to +10 mV (Na^v1.8) or −10 mV (TTX-s Na^v). Peak currents of the test pulse were measured, normalized (I  ^Na/I  ^Namax), plotted against the prepulse potential, and fitted with a Boltzmann function. Decay time constants of peak I  ^Nawere obtained from a monoexponential fit to the decay phase of the macroscopic Na⁺current from 90% of peak current. Use-dependent block for Na^v1.8 was studied at 1, 3, and 10 Hz with 60 10-ms test pulses up to a final potential of +10 mV. Peak currents were measured, normalized to the first pulse and plotted against pulse number. IC⁵⁰values were determined by least squares fitting of data to the Hill equation: Y = 1/(1 + 10((logIC⁵⁰− X)^h)), where Y is the current amplitude, X is the isoflurane concentration, and h is Hill slope. Statistical significance was assessed by analysis of variance with Newman-Keuls post hoc  test, or paired or unpaired Student t  test. P < 0.05 was considered statistically significant. The programs used for data acquisition and analysis were pClamp 10 (Axon/Molecular Devices), Excel (Microsoft Inc., Redmond, WA), and Prism 5 (GraphPad Software Inc., San Diego, CA). Values are reported as mean ± SEM unless otherwise stated.

ND7/23 cells express endogenous TTX-s Na⁺currents (I  ^Na) with properties similar to TTX-s currents in isolated DRG neurons.27Though the specific Na⁺channel isoforms responsible for TTX-s currents in ND7/23 cells are unknown, DRG neurons express a mixed population of Na⁺channels that includes Na^v1.1, Na^v1.2, Na^v1.6, and Na^v1.7.5,6,33We compared the effects of isoflurane on endogenously expressed TTX-s I  ^Nain untransfected ND7/23 cells and on TTX-r I  ^Nain ND7/23 cells transiently transfected with rat Na^v1.8 in the presence of 300 nm tetrodotoxin to block endogenous TTX-s I  ^Na, since Na^v1.8 is resistant to tetrodotoxin (IC⁵⁰> 100 μm).3ND7/23 cells expressed voltage-gated TTX-s I  ^Na(−1,620 ± 880 pA at V^h=−70 mV, n = 6; −2,220 ± 680 pA at V^h=−140 mV, n = 6, mean ± SD) that rapidly activated and inactivated upon depolarization (fig. 1A, left). These Na⁺currents were completely inhibited by 300 nm tetrodotoxin, indicating that ND7/23 cells do not express detectable endogenous TTX-r Na⁺channels (fig. 1A, right). ND7/23 cells transfected with Na^v1.8 α-subunit showed prominent voltage-gated TTX-r I  ^Na(−1,830 ± 840 pA at V^h=−70 mV, n = 8; −1,920 ± 620 pA at V^h=−140 mV, n = 8, mean ± SD) in the presence of 300 nm tetrodotoxin (fig. 1B).30Transfection of EGFP alone did not result in expression of TTX-r I  ^Na(data not shown).

Current-voltage relationships were determined for TTX-r Na^v1.8 and TTX-s Na^vat the physiologic holding potential of −70 mV (fig. 2). Peak I  ^Nawas activated at a command potential of +10 mV for TTX-r Na^v1.8 and −10 mV for TTX-s Na^v. At a concentration of isoflurane (0.53 ± 0.06 mm) equivalent to 1.8 minimal alveolar concentration (MAC) in rat after temperature correction to 24°C,34,35the normalized peak I  ^Nawas 0.55 ± 0.03 for TTX-r Na^v1.8 (n = 8) and 0.56 ± 0.06 for TTX-s Na^v(n = 6) (fig. 2A). Inhibition was reversible upon washout of isoflurane (data not shown). Normalized conductance (G /G  ^max) plotted against command potential indicated no significant effects of isoflurane on voltage-dependence of activation for TTX-r Na^v1.8 or TTX-s Na⁺currents (fig. 2B, table 1). From a more hyperpolarized holding potential of −140 mV, at which most channels are in the closed resting state, isoflurane was much less effective and reduced normalized peak I  ^Nato 0.91 ± 0.03 for TTX-r Na^v1.8 (n = 8) and to 0.91 ± 0.01 for TTX-s Na^v(n = 6) (fig. 3). There was no significant effect of isoflurane on the voltage-dependence of activation of TTX-r Na^v1.8 or TTX-s Na^vfrom either holding potential (for V^1/2and k  values, see table 1). Further analysis of time-to-peak values, the interval from the beginning of the test pulse to peak I  ^Naamplitude, also showed no significant difference between control and isoflurane for TTX-r Na^v1.8 and TTX-s Na^v(data not shown).

The voltage-dependence of Na⁺channel inactivation was studied by using a 2-pulse protocol that consisted of a series of command potentials from −120 mV to +20 mV in 10-mV steps, followed by a test pulse to elicit peak I  ^Na(+10 mV for TTX-r Na^v1.8 and −10 mV for TTX-s Na^v). This standard approach assesses the fraction of channels available for activation by the second pulse. As the membrane potential of the prepulse becomes more positive, more channels enter inactivated and nonconducting states such that fewer channels are available for activation by the second test pulse. Inactivation curves were determined by using two different prepulse durations; fast inactivation was measured by using a 15-ms prepulse27and steady-state inactivation using a 500-ms prepulse (fig. 4). The I  ^Naof the second test pulse was normalized to peak I  ^Na(I  ^Na/I  ^Namax), plotted against prepulse potential, and fitted to a standard Boltzmann function from which the voltage of half-inactivation (V^1/2) was determined (table 1). Isoflurane (0.53 ± 0.06 mm) produced a negative shift in the voltage-dependence of steady-state inactivation (500 ms prepulse) of −5.6 ± 0.8 mV for TTX-r Na^v1.8 and −7.1 ± 0.3 mV for TTX-s Na^v(P < 0.001), and a negative shift in the voltage-dependence of fast inactivation (15-ms prepulse) of −9.2 ± 2.1 mV for TTX-r Na^v1.8 and −15 ± 2.3 mV for TTX-s Na^v(P < 0.01). There were no significant effects on slope values. The time constant of Na⁺current decay at peak I  ^Na(τ) was significantly increased by isoflurane for both TTX-r Na^v1.8 and TTX-s Na^v. The τ values for Na^v1.8 were 2.5 ± 0.1 ms in control and 3.0 ± 0.2 ms with isoflurane (P < 0.05, n = 8), and for TTX-s Na^vwere 0.54 ± 0.04 ms in control and 0.58 ± 0.04 ms with isoflurane (P < 0.05, n = 6).

IC⁵⁰values for isoflurane inhibition of I  ^Nawere obtained by eliciting peak I  ^Nafrom a holding potential of −70 mV. Normalized peak I  ^Navalues were fitted to the Hill equation to yield IC⁵⁰and Hill slope values (fig. 5). The IC⁵⁰values of 0.67 ± 0.06 mm for TTX-r Na^v1.8 and 0.66 ± 0.09 mm for TTX-s Na^v, and Hill slopes of −1.12 ± 0.16 for TTX-r Na^v1.8 and −0.85 ± 0.14 for TTX-s Na^vwere not significantly different. Significant inhibition occurred at isoflurane concentrations as low as 0.17 ± 0.01 mm (equivalent to 0.58 MAC after temperature correction to 24°C) for both TTX-r Na^v1.8 (P < 0.01; n = 6) and TTX-s Na^v(P < 0.001; n = 8).

At the physiologic holding potential of −70 mV, isoflurane (0.53 ± 0.05 mm) significantly reduced the normalized peak I  ^Nato 0.55 ± 0.03 for TTX-r Na^v1.8 (P < 0.001, n = 8) and to 0.56 ± 0.06 for TTX-s Na^v(P < 0.01, n = 5) (fig. 6). Inhibition by isoflurane was significantly less from a holding potential of −140 mV, at which most channels are in the closed resting state (normalized peak I  ^Nawas 0.91 ± 0.03 (P < 0.05, n = 8) for TTX-r Na^v1.8 and 0.91 ± 0.01 (P < 0.001, n = 6) for TTX-s Na^v).

Preferential interaction of isoflurane with the inactivated state of Na⁺channels results in accumulation of drug-bound channels during high-frequency stimulation.36Native TTX-r Na⁺channels found in DRG neurons recover quickly from inactivation after membrane repolarization,37and similar behavior has been reported for Na^v1.8 in ND7/23 cells.30We used 60 pulses to +10 mV to determine the decay in peak amplitude of Na^v1.8 at various frequencies (1, 3, and 10 Hz). In control experiments, use-dependent block was more pronounced at higher stimulation frequencies (fig. 7). In the presence of isoflurane (0.56 ± 0.08 mm), Na^v1.8 currents showed a greater use-dependent decrement compared to control at all three stimulation frequencies tested. At a stimulation frequency of 1 Hz, the plateau of normalized peak I  ^Na, which was determined by fitting the data to a single exponential function, was 0.94 ± 0.01 in control and 0.85 ± 0.02 in the presence of isoflurane (P < 0.001, n = 5). At a stimulation frequency of 3 Hz, the plateau of normalized peak I  ^Nawas 0.88 ± 0.01 in control and 0.81 ± 0.01 in the presence of isoflurane (P < 0.001, n = 5). At the highest frequency of 10 Hz, the plateau of normalized peak I  ^Nawas 0.78 ± 0.01 in control and 0.64 ± 0.01 in the presence of isoflurane (P < 0.001, n = 5). Control data for use-dependent reductions in Na^v1.8 current are comparable to those published previously for this channel.27,30 

Voltage-gated Na⁺channel isoforms are pharmacologically distinguishable, and are in fact classified, by their differential sensitivities to the specific inhibitor tetrodotoxin. Considerable evidence indicates that TTX-s isoforms are reversibly inhibited by clinical concentrations of volatile anesthetics, but anesthetic effects on the TTX-r isoforms are poorly characterized. A previously published study suggested that Na^v1.8 was unique among Na^visoforms tested in its resistance to inhibition by isoflurane when tested using heterologous expression in amphibian oocytes,21but this has not been confirmed in neuronal cells. We investigated the effects of the commonly used inhaled anesthetic isoflurane on endogenously expressed TTX-s and heterologously expressed TTX-r Na^v1.8 currents in a neuronal cell line.

Isoflurane inhibited both TTX-r Na^v1.8 and endogenous TTX-s Na^vwith similar potencies (IC⁵⁰= 0.67 mm or 0.66 mm, respectively). These concentrations correspond to 2.3 MAC in rat after temperature correction to 24°C, and they are similar to those reported previously for inhibition of Na^v1.2, Na^v1.4, and Na^v1.5 heterologously expressed in Chinese hamster ovary cells by isoflurane (IC⁵⁰= 0.70, 0.61, and 0.45 mm, respectively).20Although the IC⁵⁰values are somewhat higher than clinically relevant concentrations, significant inhibition occurs in the more clinically relevant concentration range of more than 0.5 times MAC.20,24,38Moreover, small reductions in I  ^Nacan have large physiologic effects due to nonlinear coupling.24The finding that isoflurane inhibits Na^v1.8 expressed in a mammalian neuronal cell line but not when expressed in Xenopus  oocytes21demonstrates the importance of an appropriate expression system for pharmacological studies of these channels.

It is now clear that both TTX-r and TTX-s Na^visoforms are inhibited by inhaled anesthetics and do not exhibit major differences in anesthetic sensitivity.20Sensitivity to inhaled anesthetics is even present in the homologous prokaryotic Na⁺channel NaChBac, indicating that anesthetic sensitivity is related to a fundamental evolutionarily conserved Na⁺channel property.39In addition to their differential sensitivities to tetrodotoxin, Na^visoforms have been reported to have different sensitivities to local anesthetics. In rat DRG neurons and with oocyte expression, TTX-r currents (primarily Na^v1.8) are more sensitive to inhibition by lidocaine than TTX-s channels, despite their highly conserved amino acid sequences.7,14Sensitivity of Na^visoforms to local anesthetics is determined primarily by conserved residues in the DIV-S6 segment,40whereras the greater sensitivity of Na^v1.7 and Na^v1.8 to lidocaine has been proposed to result from minor sequence differences in the DI and DII S6 segments,14although this could be affected by differences in voltage dependence of inactivation. The comparable sensitivities of various Na^visoforms to isoflurane suggest a conserved drug-binding domain, perhaps in DIV-S6.

Analysis of Na^v1.8 pharmacology has been hampered by difficulties in expressing functional channels. Initial expression in Xenopus  oocytes showed relatively small currents,4and attempts by other groups to express Na^v1.8 in mammalian cell lines, including COS-7,41CHO,42and HEK-293 cells,27resulted in very low levels of functional expression. However ND7/23 cells, derived from rat DRG and mouse neuroblastoma (N18TG2) cells, are suitable for transient and stable expression of recombinant Na^v1.8 in a mammalian neuronal environment.28These cells endogenously express Na^vβ1- and β3-subunits,27which are sufficient for the functional expression and stability of Na^v1.8 α-subunits. Cotransfection of Na^v1.8 with the β1-subunit29or β3-subunit27does not alter current kinetics, activation, or inactivation characteristics of TTX-r currents in ND7/23 cells.

Isoflurane had negligible effects on the voltage dependence of Na^v1.8 activation, but it produced a hyperpolarizing shift in the voltage-dependence of fast and steady-state inactivation. This behavior is consistent with selective interaction of isoflurane with channels in the inactivated state as described previously for other Na⁺channel isoforms including Na^v1.2 and Na^v1.4.19–21The molecular basis of this block has yet to be determined for volatile anesthetics. Our results suggest that the shift in voltage-dependence of inactivation might result from slowing of inactivation evidenced by the increased time constants of current decay. The functional consequence is a reduction in the range of membrane potentials over which Na^v1.8 can operate, as confirmed by the voltage-dependence of isoflurane inhibition.

Other Na⁺channel blockers such as local anesthetics (e.g. , lidocaine) and certain anticonvulsants and antiarrhythmics also exhibit state-dependent drug interactions with Na^vas described by the modulated receptor hypothesis.43Voltage-dependent block by local anesthetics results in a hyperpolarizing shift in steady-state inactivation, thus enhancing channel block at normal as opposed to hyperpolarized potentials. Isoflurane apparently inhibits Na⁺channels by a similar mechanism involving enhanced inactivation. Selective interaction with inactivated states is consistent with the use-dependent block by isoflurane, which increases the fraction of channels in the inactivated state. Na⁺channels undergo both fast and slow inactivation, and slow inactivation contributes to the use-dependent effects of some drugs.44The contribution of slow inactivation to the effects general anesthetics on Na^vblock is an interesting question for future investigation.

The rate at which Na⁺channels recover from inactivation (repriming) determines how well channels respond to high firing rates. Isoform-specific differences in repriming rates have been reported.45Interestingly, repriming rates of TTX-r Na⁺currents, which are “slow” in terms of time to peak current and time constant of current decay, are about 10-fold faster than those of TTX-s Na⁺currents in rat DRG neurons. Use-dependent block of Na^v1.8 by isoflurane could be due to its slow dissociation from blocked channels during repolarization, effectively slowing the repriming rate, but this is unlikely given the low affinity interaction. Lidocaine does not interfere with movement of the cytoplasmic inactivation loop, which is the underlying mechanism for fast inactivation, such that lidocaine-induced slowing of Na⁺channel repriming does not result from slow recovery of the fast-inactivation gate.46This suggests that use-dependent block does not involve accumulation of fast-inactivated channels, but it could involve effects on slow inactivation mechanisms. By analogy with local anesthetics, stabilization of inactivated channel states and/or open channel block by isoflurane is currently a more plausible explanation.38 

The Na⁺current underlying the depolarization phase of the action potential in nociceptive DRG neurons is carried primarily by Na^v1.8, which is expressed exclusively in this cell type.4,10Slowly inactivating TTX-r Na⁺currents are eliminated in DRG neurons of Na^v1.8 knockout mice, which confirms the role of Na^v1.8 in conducting these currents.8Both antisense and knock-out studies support a role for Na^v1.8 activation in inflammatory pain.8Previous studies using antisense nucleotides suggested a role for Na^v1.8 in neuropathic pain,13but a recent study shows that Na^v1.8 is necessary for mechanical, cold, and inflammatory pain, but not for neuropathic and heat pain.47Visceral pain, a major consideration in the perioperative setting, has been attributed to Na^v1.8 since knockout mice show decreased visceral pain and referred hyperalgesia.48Subanesthetic concentrations of isoflurane, which would probably have relatively small effects on Na^v1.8, depress the nociceptive reflex to single electrical stimuli in humans,49,50whereas anesthetic concentrations of 1 MAC are required to depress the response to repetitive stimuli critical to central hyperexcitability in humans.49,50In addition, volatile anesthetics, including isoflurane, significantly suppress development of spinal sensitization in the rat paw formalin test, which has implications for the development of postoperative pain.51Moreover, isoflurane has peripheral antinociceptive effects in a number of animal models in which supraspinal modulatory and/or pronociceptive effects were surgically or pharmacologically eliminated.52–54The anesthetic concentrations required for these effects on pain processing are consistent with the sensitivity of Na^v1.8 to inhibition by isoflurane and a possible role in nociceptive processing by DRG neurons. This inhibition would be enhanced at high firing frequencies and depolarized membrane potentials, conditions that occur with tissue injury and inflammation, based on the frequency- and voltage-dependence of isoflurane block. Recent studies also implicate volatile anesthetic activation and sensitization of TRPV1 ion channels in lowering the threshold for heat activation.55Anesthetic modulation of peripheral Na⁺channels such as Na^v1.8, therefore, has the potential to modulate these poorly characterized pronociceptive mechanisms. The role of isoflurane inhibition of Na^v1.8 in acute perioperative pain and the development of hyperexcitability is an interesting topic for further investigation.

In conclusion, both TTX-r Na^v1.8 and TTX-s Na^vwere inhibited by isoflurane at concentrations that occur during clinical anesthesia. This is consistent with a conserved drug-binding site among various Na^visoforms. The critical role of Na^v1.8 in peripheral pain mechanisms suggests that its inhibition could contribute to the antinociceptive and possibly antiinflammatory effects of isoflurane and other inhaled anesthetics capable of blocking these channels.