n-Alcohols inhibit voltage-gated Na+ channels expressed in Xenopus oocytes

Abstract

Voltage-gated sodium channels are essential for the initiation and propagation of action potentials in excitable cells and are known as a target of local anesthetics. In addition, inhibition of sodium channels by volatile anesthetics has been proposed as a mechanism of general anesthesia. The n-alcohols produce anesthesia, and their potency increases with carbon number until a "cut-off" is reached. In this study, we examined effects of a range of n-alcohols on Na(v)1.2 subunits to determine the alcohol cut-off for this channel. We also studied the effect of a short-chain alcohol (ethanol) and a long-chain alcohol (octanol) on Na(v)1.2, Na(v)1.4, Na(v)1.6, and Na(v)1.8 subunits, and we investigated the effects of alcohol on channel kinetics. Ethanol and octanol inhibited sodium currents of all subunits, and the inhibition of the Na(v)1.2 channel by n-alcohols indicated a cut-off at nonanol. Ethanol and octanol produced open-channel block, which was more pronounced for Na(v)1.8 than for the other sodium channels. Inhibition of Na(v)1.2 was due to decreased activation and increased inactivation. These results suggest that sodium channels may have a hydrophobic binding site for n-alcohols and demonstrate the differences in the kinetic mechanisms of inhibition for n-alcohols and inhaled anesthetics.

Figure 1

Inhibitory effects of ethanol (C2) (190 mM) and octanol (C8) (0.057 mM) on sodium channels at different holding potentials. (A) Traces of sodium currents evoked by a 50-ms depolarizing pulse to −20 mV from a holding potential of −90 mV (V_max) and to −20 mV from a holding potential which induced half maximal current (V_1/2), in the absence and presence of ethanol in an oocyte expressing Na_v1.2. (B) Time course of ethanol effects on sodium currents of Na_v1.2. Currents were elicited by 50-ms depolarizing pulses to −20 mV applied every 10 s from a V_1/2 holding potential. The current is normalized to the initial values. Filled circles represent control and washout, and open circles represent currents during ethanol treatment. Ethanol was applied for 3 min. (C) Traces of sodium currents evoked by a 50-ms depolarizing pulse to −20 mV from V_max and V_1/2 in the absence and presence of octanol in an oocyte expressing Na_v1.2. (D) Time course of octanol effects on sodium currents of Na_v1.2. (E) Percent inhibition of sodium current by ethanol and octanol in oocytes expressing Na_v1.2, Na_v1.4, Na_v1.6, and Na_v1.8. Open columns indicate the effect at V_max, and closed columns indicate the effect at V_1/2. V_1/2 value of Na_v1.2, Na_v1.4, Na_v1.6, and Na_v1.8 were 55.8 ± 0.5, 57.5 ± 2.0, 59.3 ± 0.4 and 43.9 ± 1.4 mV, respectively. Data are mean ± S.E.M. (n = 4–6). Differences between V_max and V_1/2 for each condition are indicated as *, p < 0.05; **, p < 0.01; and ***, p < 0.001 (one-way ANOVA).

Figure 2

Effects of n-alcohols on sodium currents in oocytes expressing Na_v1.2. Concentration-response curves for n-alcohols (methanol to dodecanol) on sodium currents elicited by a 50-ms depolarizing pulse to −20 mV from V_1/2 holding potential. V_1/2 value of Na_v1.2 was 54.7 ± 0.3 mV. Data are represented as means ± S.E.M. (n = 5–6). The data were fit by a logistic equation to the give IC₅₀s and Hill slopes shown in Table 1.

Figure 3

Effects of ethanol (190 mM) and octanol (0.057 mM) on I–V curves of sodium currents in oocytes expressing Na_v1.2 and Na_v1.8. (A) Representative I_Na traces from oocytes expressing Na_v1.2 in the absence and presence of ethanol or octanol. Currents were elicited by 50-ms depolarizing steps between −80 and 50 mV in 10-m V increments from holding potentials of −90 mV. (B) Effects of ethanol and octanol on representative I–V curves elicited from V_max holding potential for Na_v1.2. (C) Effects of ethanol and octanol on representative I–V curves elicited from V_1/2 holding potential in oocytes expressing Na_v1.2. (D) Representative I_Na traces in oocytes expressing Na_v1.8 in the absence and presence of ethanol and octanol. (E) Effects of ethanol and octanol on representative I–V curves elicited from a V_max holding potential in oocyte expressing Na_v1.8. (F) Effects of ethanol and octanol on representative I–V curves elicited from a V_1/2 holding potential in oocyte expressing Na_v1.8.

Figure 4

Effects of ethanol (190 mM) and octanol (0.057 mM) on channel activation in oocytes expressing Na_v1.2, Na_v1.4, Na_v1.6, and Na_v1.8 from V_max holding potential (A panels) or V_1/2 holding potential (B panels). V_1/2 value of Na_v1.2, Na_v1.4, Na_v1.6, and Na_v1.8 were 54.0 ± 0.9, 55.3 ± 1.2, 58.7 ± 0.9 and 42.0 ± 1.2 mV, respectively. Data are shown as mean ± S.E.M. (n = 4–6). Activation curves fitted to a Boltzmann equation and V_1/2 are shown in Table 2.

Figure 5

Effects of ethanol and octanol on inactivation curves in oocytes expressing Na_v1.2 (A), Na_v1.4 (B), Na_v1.6 (C), and Na_v1.8 (D). Currents were elicited by a 50-ms test pulse to −20 mV (Na_v1.8: 10 mV) after 200-ms prepulses (Na_v1.8: 500-ms) ranging from −140 to 0 mV in 10-mV increments. Data shown as mean ± S.E.M. (n = 4–6). Inactivation curves were fitted to a Boltzmann equation and the V_1/2 values are shown in Table 2.

Figure 6

Sequence alignment of Na_v1.2, Na_v1.4, Na_v1.6, and Na_v1.8 for segment 5 in domain I, segment 5 in domain II, and segment 6 in domain II. Three amino acids were selected in these areas because these amino acids are different in Na_v1.8 from those in Na_v1.2, Na_v1.4, and Na_v1.6 and are in trans-membrane regions close to the extracellular surface. The amino acids of Na_v1.8 were introduced into Na_v1.2 and Na_v1.4, giving the single mutants: M271K, M900K, and M965T (Na_v1.2 mutants), and M273K, M719K, and M784T (Na_v1.4 mutants).

Figure 7

The inhibitory effect of ethanol (190 mM), hexanol (0.57 mM), and octanol (0.057 mM) on Na_v1.2 wild type and three mutants. Currents were elicited by a 50-ms depolarizing pulse to −20 mV applied every 10 s from V_max and V_1/2 holding potential, and each alcohol was applied for 3 min. V_1/2 value of Na_v1.2 WT, M271K, M900K, and M965T were 51.0 ± 1.0, 43.8 ± 0.6, 56.2 ± 1.1 and 48.6 ± 0.9 mV, respectively. Data are mean ± S.E.M. (n = 4–6). **, p < 0.05 by one-way ANOVA.

Figure 8

Comparison of IC₅₀ (EC₅₀) and ΔG values for n-alcohols between published studies and data from the present study. (A) Relationship between the logarithms of IC₅₀ and carbon number. Filled circles, EC₅₀s for tadpole anesthesia (Alifimoff et al., 1989); open circles, IC₅₀s for Na_v1.2 (present study); open triangles, IC₅₀s for squid axon sodium channel (Haydon and Urban, 1983); crosses, IC₅₀s for NMDA receptors (Peoples and Weight, 1995). (B) Gibb’s free energy change for partitioning from the water phase to the site of action. The free energy change contributed by each methylene group (ΔΔG) was calculated from slope of Figure 8B and is given in Table 3.