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NEUROPHARMACOLOGY
Waggoner Center for Alcohol and Addiction Research, The University of Texas at Austin, Austin, Texas
Received February 25, 2008; accepted April 22, 2008.
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Abstract |
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; Mihic et al., 1997), inhibits glutamate receptor function (Wong et al., 1998), and potentiates neuronal nicotinic acetylcholine receptor function (Godden et al., 2001). However, it is not known whether actions of alcohols on these channels are sufficient to explain the behavioral effects of the n-alcohols.
Voltage-gated sodium channels play an essential role in action potential initiation and propagation in excitable cells of nerve and muscle. Nine distinct pore-forming 
-subunits, which are associated with auxiliary β-subunits, have been identified (Catterall, 2000; Catterall et al., 2005). Each -subunit has a different pattern of development and localization and demonstrates subtle differences in electrophysiological characteristics (Goldin, 2001). Moreover, these subunits have distinct physiological and pathophysiological roles. For example, multiple mutations of Nav1.1 can cause epilepsy (Escayg et al., 2001), and mutations of Nav1.5 are associated with arrhythmia, Brugada syndrome (Cordeiro et al., 2006), and long-QT syndrome (Vatta et al., 2006). The sodium channels expressed in dorsal root ganglion (DRG) (Nav1.7, Nav1.8, and Nav1.9) play an important role in pain (Wood et al., 2004; Cummins et al., 2007).
Recent studies have shown that volatile anesthetics inhibit sodium channel function at clinically relevant concentrations in some neurons (Ratnakumari and Hemmings, 1998; Ouyang et al., 2003), and in some recombinant mammalian sodium channels (Rehberg et al., 1996; Shiraishi and Harris, 2004; OuYang and Hemmings, 2007), suggesting sodium channels as a potential target for inhaled anesthetics. n-Alcohols can produce a state of general anesthesia, historically ethanol has been used for this purpose (Dundee et al., 1969), and there is evidence that ethanol and other alcohols inhibit sodium channel function. For example, a high concentration of ethanol (500 mM) inhibited sodium influx in synaptosomes isolated from rodent brain (Harris and Bruno, 1985), and Wu and Kendig (1998) showed that tetrodotoxin (TTX)-resistant and TTX-sensitive sodium channels in rat DRG neurons are inhibited by ethanol (50–200 mM) (Wu and Kendig, 1998). Haydon and Urban (1983) found that longer chain n-alcohols (pentanol to decanol) inhibited the sodium current of the squid giant axon (Haydon and Urban, 1983). Some ion channels are affected by short, but not long, chain length n-alcohols, and this alcohol "cut-off" has proven useful in characterizing alcohol action on ion channels (Wick et al., 1998). To extend these studies to sodium channels, we explored the effects of a series of n-alcohols on sodium channels.
Nav1.1, Nav1.2, Nav1.3, and Nav1.7 are TTX-sensitive and closely related by phylogenetic analysis, and all of their genes are located on human chromosome 2q23–24. Nav1.5, Nav1.8, and Nav1.9 are TTX-resistant and also closely related, and their genes are located on chromosome 3p21–24. Nav1.4 and Nav1.6 are phylogenetically distant from the other two clusters. We selected four of these subunits for study, as follows. We examined Nav1.2, which is expressed primarily in the central nervous system; Nav1.4, which is expressed primarily in skeletal muscle; Nav1.6, which is expressed primarily in the central nervous system and DRG; and Nav1.8, which is expressed in DRG.
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Materials and Methods |
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-subunit (a gift from Dr. W. A. Catterall, University of Washington, Seattle, WA), cDNA for human Nav1.4 -subunit (a gift from Dr. A. L. George, Vanderbilt University, Nashville, TN), cDNA for rat Nav1.6 -subunit (a gift from Dr. A. L. Goldin, University of California, Irvine, CA), cDNA for rat Nav1.8 -subunit (a gift from Dr. A. N. Akopian, University of Texas Health Science Center, San Antonio, TX), and cDNA for human β1-subunit (a gift from Dr. A. L. George, Vanderbilt University).
Site-Directed Mutagenesis. Mutations were created using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). In brief, polymerase chain reaction was performed using the cDNA of wild-type Nav1.2 and Nav1.4 -subunit clone and the mutagenesis primers designed with the desired mutation at the appropriate place. Then, Escherichia coli-competent cells (XL1-Blue or One-shot TOP10/P3) were transformed with the polymerase chain reaction product digested with DpnI.
cRNA Preparation and Oocyte Injection. cDNA was linearized with ClaI (Nav1.2 -subunit), EcoRI (Nav1.4 -subunit, β1-subunit), and NotI (Nav1.6 -subunit), XbaI (Nav1.8 -subunit) cRNAs and transcribed using T6 (Nav1.4, 1.8 ,β1-subunit) or T7 (Nav1.2, 1.6 -subunit) RNA polymerase of mMESSAGE mMACHINE kit (Ambion, Austin, TX). Preparation of X. laevis oocytes and microinjection of the cRNA were performed as described previously (Shiraishi and Harris, 2004). In brief, stage IV–VI oocytes were manually isolated from a removed portion of ovary. The oocytes were then treated with collagenase (0.5 mg/ml) for 10 min and placed 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, and 0.91 mM CaCl2, adjusted to pH 7.5], supplemented with 10,000 units of penicillin, 50 mg of gentamicin, 90 mg of theophylline, and 220 mg of sodium pyruvate per liter (incubation medium). Nav -subunit cRNAs were coinjected with β1-subunit cRNA at a ratio of 1:10 (total volume was 20
40 ng/50 nl) into Xenopus oocytes (all -subunits were coinjected with β1-subunit). The injected oocytes were incubated at 19°C in incubation medium, and 2 to 6 days after injection, they were used in electrophysiological recordings.
Electrophysiological Recordings. All electrical recording was performed at room temperature (23°C). The oocytes were placed in a rectangular chamber (approximately 100-µl volume) and perfused at 2 ml/min with frog Ringer's solution containing 115 mM NaCl, 2.5 mM KCl, 10 mM HEPES, and 1.8 mM CaCl2 at pH 7.2 using a peristaltic pump (Cole-Palmer Instrument Co., Chicago, IL). Recording electrodes were prepared with borosilicate glass using a puller (P-97; Sutter Instruments Company, Novato, CA). Microelectrodes were filled with 3 M KCl/0.5% low-melting-point agarose, and they had resistances between 0.3 and 0.5 M
. The whole-cell voltage clamp was achieved through these two electrodes using a Warner oocyte clamp (model OC-725C; Warner Instruments, Hamden, CT).
The amplitude of expressed sodium currents was typically 2 to 15 µA, and currents were recorded and analyzed using pCLAMP 7.0 software (Axon Instruments, Foster City, CA). The transients and leak currents were subtracted using the P/N procedure. Capacitance and 60 to 80% series resistance were compensated, and leak current was subtracted using P/4 protocols (Shiraishi and Harris, 2004). For the poorly water-soluble alcohols (heptanol to dodecanol), stocks were prepared in dimethyl sulfoxide and diluted and sonicated in frog Ringer's solution to a final dimethyl sulfoxide concentration not exceeding 0.05%. The n-alcohols were then perfused for 3 min to reach equilibrium. The loss of concentration from vial to bath was approximately 50 to 70% for long-chain alcohols from heptanol to dodecanol (Dildy-Mayfield et al., 1996).
The voltage dependence of activation was determined by 50-ms depolarizing pulses from a holding potential of –90 mV to 50 mV in 10-mV increments or from a holding potential causing half-maximal current (V1/2) (approximately from –50 mV to –60 mV) to 50 mV in 10-mV increments. Activation curves 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, and k is the slope factor. The G value for each oocyte was calculated using the formula 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.
Data Analysis. All values are presented as the mean ± S.E.M. The n values refer to the number of oocytes studied. Each experiment was performed with oocytes from at least two different frogs. Statistical analyses were performed using a one-way analysis of variance (ANOVA) for multiple comparisons and a t test using GraphPad Prism software (GraphPad Software Inc., San Diego, CA). We also calculated Hill slope and IC50 values using GraphPad Prism.
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). Currents were elicited by a 50-ms depolarizing pulse to –20 mV applied every 10 s from –90 mV (Vmax) or a holding potential of V1/2. It was found that ethanol inhibited INa induced by Nav1.2 at Vmax, and the inhibitory effect of ethanol was more potent at V1/2 (Fig. 1, A and B). Octanol also inhibited INa induced by Nav1.2 at both Vmax and V1/2, but more effectively at V1/2 (Fig. 1, C and D). Ethanol and octanol were also tested on the other -subunits: Nav1.4, Nav1.6, or Nav1.8. Ethanol reduced the peak INa induced by Nav1.2, Nav1.4, Nav1.6, and Nav1.8 by 19 ± 1, 19 ± 4, 14 ± 1, and 28 ± 1% at Vmax, respectively, and 29 ± 1, 35 ± 2, 25 ± 1, and 30 ± 3% at V1/2, respectively (Fig. 1E). Octanol reduced the peak INa induced by Nav1.2, Nav1.4, Nav1.6, and Nav1.8 by 17 ± 1, 19 ± 3, 17 ± 1, and 13 ± 1% at Vmax, respectively, and 36 ± 2, 46 ± 2, 38 ± 4, and 19 ± 1% at V1/2, respectively (Fig. 1E). Thus, for Nav1.2, Nav1.4, and Nav1.6, ethanol and octanol inhibited the peak INa more effectively at V1/2 than at Vmax. In contrast, for Nav1.8, ethanol similarly suppressed the peak INa at V1/2 and Vmax (Fig. 1E).
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Effects of n-Alcohols on Nav1.2 Currents. Increases in carbon chain length shifted the concentration-response curves to lower alcohol concentrations, but these shifts became very small as the chain length increased from octanol to nonanol to decanol, indicating a cut-off approximately at nonanol (Fig. 2). The IC50 values and slopes calculated from dose-response data are shown in Table 1. The IC50 value for dodecanol was estimated by extrapolation because the inhibitory effect at highest concentration of dodecanol was less than 50%.
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Effects of Ethanol and Octanol on Activation and Inactivation of Sodium Currents. The voltage dependence of activation was determined by 50-ms depolarizing pulses from a holding potential of –90 mV (Vmax) to 50 mV in 10-mV increments or from a holding potential of V1/2 to 50 mV in 10-mV increments for Nav1.2, Nav1.4, Nav1.6, or Nav1.8. The activation curves were derived from the I-V curves (see Materials and Methods), and the peak INa was reduced by ethanol (190 mM) and octanol (0.057 mM) at Vmax and V1/2 holding potentials with all subunits (Fig. 3). Ethanol and octanol shifted the midpoint of steady-state activation in a depolarizing direction by 2.7 and 3.3 mV, respectively, at Vmax for Nav1.2 (Fig. 4A; Table 2). These changes were small but significant statistically.
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The effect of ethanol and octanol on the steady-state inactivation was also investigated. Ethanol weakly (and insignificantly) shifted the V1/2 in the hyperpolarizing direction by 1.0, 2.2, 1.2, and 2.1 mV in Nav1.2, Nav1.4, Nav1.6, and Nav1.8, respectively (Fig. 5; Table 2). Alternatively, octanol shifted the V1/2 in the hyperpolarizing direction significantly by 3.4, 3.6, and 3.5 mV in Nav1.2, Nav1.4, and Nav1.6, respectively, although the shift with Nav1.8 was not significant (1.7 mV) (Fig. 5; Table 2). On the whole, octanol was more effective than ethanol with the steady-state inactivation of Nav1.2, Nav1.4, or Nav1.6, but the effect of octanol on Nav1.8 was smaller than the effect of ethanol.
Effects of Ethanol and Octanol on Mutated Nav1.2 and Nav1.4 Channels. We found that the inhibitory effect of ethanol on Nav1.8 was similar at Vmax and at V1/2, but it was greater at V1/2 than at Vmax for Nav1.2, Nav1.4, and Nav1.6 (Fig. 1E). To explore the molecular differences that might account for this distinction between Nav1.8 and other -subunits, we introduced single-point mutants into Nav1.2 and Nav1.4 to see whether these mutations would switch the alcohol sensitivity of these subunits. We selected three amino acids that were different in Nav1.8 from Nav1.2, Nav1.4, and Nav1.6 in domains I and II (Fig. 6), and we hypothesized that these amino acid differences could be functionally important because of the polarity differences of these amino acids between Nav1.8 and other -subunits. We introduced the amino acids found in Nav1.8 into Nav1.2 and Nav1.4 (M271K, M900K, and M965T in Nav1.2 and M273K, M719K, and M784T in Nav1.4). At V1/2, the mutations in Nav1.2 did not alter the inhibitory effect of ethanol, hexanol, and octanol, but at Vmax the M900K mutant increased the inhibitory effect of these alcohols significantly compared with Nav1.2 wild type (Fig. 7). Ethanol inhibited Nav1.2 wild type and M900K by 14 ± 1 and 20 ± 2%, respectively; hexanol inhibited Nav1.2 wild type and M900K by 7 ± 1 and 13 ± 2%, respectively; and octanol inhibited Nav1.2 wild type and M900K by 11 ± 1 and 16 ± 2%, respectively. There were no differences in alcohol inhibition between Nav1.4 wild type and its mutants at Vmax or V1/2, although at Vmax the inhibitory effect of these three alcohols on M719K (equivalent to M900K of Nav1.2) increased slightly (data not shown).
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Discussion |
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-subunits by 30 to 40% at V1/2 and by 13 to 30% at Vmax. This is consistent with previous studies of other anesthetics. For example, anesthetic concentrations (0.25–0.4 mM) of isoflurane inhibited INa in isolated rat neurohypophysial nerve terminal by around 40% (Ouyang et al., 2003); suppressed sodium currents of Nav1.2 in Chinese hamster ovary cells (Rehberg et al., 1996); and inhibited currents from Nav1.2, Nav1.4, and Nav1.6 by approximately 30% in an oocyte expression system (Shiraishi and Harris, 2004).
Two questions that remain are 1) how much inhibition of sodium currents is required to alter neuronal function, and 2) can the observed alcohol effects contribute to anesthesia? Scholz and colleagues (Scholz et al., 1998a,b; Scholz and Vogel, 2000) demonstrated that firing frequency was reduced from 35 Hz to 21 Hz by 30 µM lidocaine in DRG neurons. They also found that the number of action potentials decreased from 21 to 10 in 750 ms by 10% inhibition of sodium currents, suggesting that even 10% inhibition may reduce action potential propagation. These previous reports, together with our present results, support the idea that alcohol actions on sodium channels could reduce neuronal firing and potentially result in anesthesia.
n-Alcohol Cut-Off and Hydrophobic Binding Sites. Alifimoff et al. (1989) demonstrated a cut-off at approximately C13 for anesthesia using tadpoles, and this raised the possibility that n-alcohols produce anesthesia by acting on a protein cavity that can accommodate as large as C12, but not larger (Alifimoff et al., 1989). One of the first proteins systematically studied with the n-alcohols was firefly luciferase. Franks and Lieb (1984, 2004) showed a remarkable correlation between luciferase inhibition and anesthetic potency (Franks and Lieb, 1984, 2004), and they suggested the existence of a binding pocket based on the n-alcohol cut-off of luciferase (Franks and Lieb, 1985). More recent studies have reported alcohol cut-offs for ligand-gated ion channels. For example, cut-offs of GABA rho, NMDA, GABAA, glycine, and neuronal nicotinic acetylcholine receptors are C7, C8, C10, C10, and C12, respectively (Peoples and Weight, 1995; Dildy-Mayfield et al., 1996; Wick et al., 1998; Zuo et al., 2001). The sodium channels show a cut-off at C9 in the present study, yet tadpoles are sensitive to C12 (and rats to at least C10; Dildy-Mayfield et al., 1996), raising the question of which of these channels is relevant, or clearly not relevant, for the anesthetic actions of long-chain alcohols. We propose that the cut-offs for GABA rho and NMDA are probably too short, but it is difficult to absolutely exclude GABAA, glycine, and sodium channels based on the cut-off.
Testing a series of n-alcohols also allows estimation of the free energy change that occurs upon transfer of the alcohol from water to its site of action. This is commonly calculated as the free energy change obtained by adding each CH2 group, which gives a measure of the hydrophobicity of the site of action. For our sodium channel, we calculated a value of 
G =–835 cal/mol/CH2 =–3.49 kJ/mol/CH2. We compared G from our study with results from other studies of n-alcohols (Fig. 8B; Table 3). Nav1.2 is similar to tadpole anesthesia in approach to cut-off and G values, although tadpole anesthesia shows a gradual change at C10–C12, whereas the sodium channel shows a more abrupt cut-off. In contrast, one ligand-gated ion channel, the NMDA receptor, shows a cut-off at much shorter chain lengths (Peoples and Weight, 1995). Thus, n-alcohol inhibition of brain sodium channel function is close to anesthetic potency in vivo, suggesting the possibility of existence that binding sites for anesthetics on sodium channels (Fig. 8A).
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Effects of Alcohol on Activation and Inactivation. The actions of ethanol and octanol on channel activation and inactivation demonstrated some common characteristics but also some differences among the four -subunits we studied. We found that suppression of sodium currents by alcohols was voltage-insensitive in the hyperpolarizing potential range and that inhibition was seen at a holding potential that produces a maximal current. These results suggest that one mechanism of alcohol action is inhibition of open channels. Although ethanol and octanol shifted the V1/2 of activation of Nav1.2 in a depolarizing direction, the shift was small (4 mV), and the V1/2 values of activations of the other subunits were not significantly changed, suggesting that alcohol effects on channel activation are not sufficient to account for inhibition of channel function. Octanol shifted the V1/2 of inactivation of the Nav1.2, Nav1.4, and Nav1.6 subunits in a hyperpolarizing direction (4 mV), and ethanol produced a small shift, which was not statistically significant. Neither ethanol nor octanol altered inactivation of Nav1.8.
From these analyses of activation and inactivation, the most consistent component of alcohol-induced suppression for the four different -subunits is suppression of open channels. For Nav1.2, Nav1.4, and Nav1.6, increased inactivation also contributes to inhibition of sodium currents for octanol, but for Nav1.8 there is only a small change in inactivation kinetics. Thus, the main component of ethanol-induced inhibition of Nav1.8 is suppression of open channels. Wu and Kendig (1998) concluded that the open state of TTX-resistant sodium channels is more sensitive to ethanol than those of TTX-sensitive sodium channels of DRG neurons (Wu and Kendig, 1998). In addition, they showed small effects of ethanol on activated state and inactivated state in TTX-sensitive and TTX-resistant sodium channels of DRG neurons (Table 4). The TTX-resistant current in DRG neurons is probably due to Nav1.8, and our results with recombinant channels are in agreement with the results from DRG neurons.
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Inhaled anesthetics also inhibit sodium channels, but the mechanism of inhibition may be different from those shown for alcohols. For example, suppression of sodium currents (human embryonic kidney 293 cells; Nav1.5) by halothane and isoflurane involves acceleration of the transition from the open to the inactivated state and stabilization of inactivated states (Stadnicka et al., 1999). Ouyang et al. (2003) suggested that general anesthetics inhibit presynaptic voltage-gated Na+ channels through enhanced inactivation (Ouyang et al., 2003). We also concluded that isoflurane stabilizes the inactivated state of several -subunits of sodium channels expressed in oocytes (Shiraishi and Harris, 2004). Compared with inhaled anesthetics, alcohols seem to be more effective as open-channel blockers, less effective at stabilizing the inactivated state, and capable of producing effects (albeit weak) on the activated state that are not found with anesthetics (Table 4).
In a recent study, OuYang and Hemmings (2007) demonstrated different mechanisms of inhibition by isoflurane for several sodium channel -subunits expressed in Chinese hamster ovary cells. They showed that isoflurane is more effective on Nav1.2 than on Nav1.4 or Nav1.5 for the resting and open states, and it is more effective on Nav1.4 or Nav1.5 than on Nav1.2 for the fast-inactivating state. That study, together with our results, indicates that -subunits differ in the mechanisms of channel inhibition by volatile anesthetics and alcohol.
Effects of Alcohol on Mutant Nav1.2. A previous report suggested that Nav1.8 was insensitive to isoflurane and concluded that one possible explanation for the insensitivity of Nav1.8 to anesthetics lies in the differences in channel gating between Nav1.8 and other channels (Shiraishi and Harris, 2004). In the present study, we found Nav1.8 to be more sensitive than other subunits to ethanol inhibition of the open state. To explore possible molecular mechanisms, we made three single mutants based on amino acid differences between Nav1.8 and other subunits. One Nav1.2 mutant, M900K, showed increased inhibition by ethanol, hexanol, and octanol at Vmax. These experiments suggest that lysine 806 of segment 5 in domain II of Nav1.8 has a role in the high sensitivity of Nav1.8 to open-channel blockage by alcohol.
Conclusions. Alcohols inhibited sodium currents induced by Nav1.2, Nav1.4, Nav1.6, and Nav1.8 at anesthetic concentrations and demonstrated a cut-off for long-chain n-alcohols. These results raise the possibility that alcohol and anesthetic inhibition of sodium channel function may be important for anesthesia, but further study is needed to clarify the relevance of sodium channels to anesthesia.
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: n-alcohol, normal alcohol; Nav, voltage-gated Na+ channel(s); DRG, dorsal root ganglion; TTX, tetrodotoxin; I-V, current voltage relationship; ANOVA, analysis of variance; INa, Na+ inward current(s); NMDA, N-methyl-D-aspartate; G, Gibb's free energy change; TTX-R, tetrodotoxin-resistant; TTX-S, tetrodotoxin-sensitive.
Address correspondence to: Dr. R. A. Harris, Waggoner Center for Alcohol and Addiction Research, The University of Texas at Austin, 1 University Station A4800, Austin, TX 78712. E-mail: harris{at}mail.utexas.edu
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