Isoflurane modulates activation and inactivation gating of the prokaryotic Na+ channel NaChBac

Abstract

Voltage-gated Na⁺ channels (Na_v) have emerged as important presynaptic targets for volatile anesthetic (VA) effects on synaptic transmission. However, the detailed biophysical mechanisms by which VAs modulate Na_v function remain unclear. VAs alter macroscopic activation and inactivation of the prokaryotic Na⁺ channel, NaChBac, which provides a useful structural and functional model of mammalian Na_v Here, we study the effects of the common general anesthetic isoflurane on NaChBac function by analyzing macroscopic Na⁺ currents (I_Na) in wild-type (WT) channels and mutants with impaired (G229A) or enhanced (G219A) inactivation. We use a previously described six-state Markov model to analyze empirical WT and mutant NaChBac channel gating data. The model reproduces the mean empirical gating manifest in I_Na time courses and optimally estimates microscopic rate constants, valences (z), and fractional electrical distances (x) of forward and backward transitions. The model also reproduces gating observed for all three channels in the absence or presence of isoflurane, providing further validation. We show using this model that isoflurane increases forward activation and inactivation rate constants at 0 mV, which are associated with estimated chemical free energy changes of approximately -0.2 and -0.7 kcal/mol, respectively. Activation is voltage dependent (z ≈ 2e₀, x ≈ 0.3), inactivation shows little voltage dependence, and isoflurane has no significant effect on either. Forward inactivation rate constants are more than 20-fold greater than backward rate constants in the absence or presence of isoflurane. These results indicate that isoflurane modulates NaChBac gating primarily by increasing forward activation and inactivation rate constants. These findings support accumulating evidence for multiple sites of anesthetic interaction with the channel.

(Scheme 1)

Figure 1.

Isoflurane effects on voltage-dependent gating. (A) Representative families of macroscopic I_Na in the absence (CTL) or presence of 0.8 mM isoflurane (ISO), elicited from HEK293FT cells expressing WT NaChBac or the mutant forms G229A and G219A as indicated. Inset shows triggering voltage-clamp protocol (stimulation frequency, 0.167 Hz; V_h, holding potential). G229A tail currents clipped for clarity. Horizontal black lines indicate baseline; calibration bars as shown. (B) Normalized I-V relationships for each channel. Currents normalized by peak current in control for each cell (n = 5–6). (C) Normalized G-V relationships in CTL and 0.8 mM ISO. Conductance (G) normalized by maximum conductance (G_max) and plotted versus voltage. Smooth lines are Boltzmann function fits to the averaged data with associated parameters (V₅₀, voltage at half amplitude; slope factor: WT, V_50(CTL) = −28.0 ± 1.0 mV, V_50(ISO) = −33.8 ± 1.5 mV, slope_CTL = 8.4 ± 0.9 mV/e, slope_ISO = 9.6 ± 1.3 mV/e; G229A, V_50(CTL) = −18.4 ± 1.3 mV, V_50(ISO) = −21.0 ± 1.2 mV, slope_CTL = 12.0 ± 1.1 mV/e, slope_ISO = 12.8 ± 1.1 mV/e; G219A, V_50(CTL) = −31.1 ± 0.5 mV, V_50(ISO) = −34.5 ± 0.7 mV, slope_CTL = 9.5 ± 0.4 mV/e, slope_ISO = 11.1 ± 0.6 mV/e). Asterisks indicate differences between CTL and ISO fits with respect to V₅₀ only (**, P < 0.01; ****, P < 0.0001; F test). Isoflurane significantly altered the slope factor of G219A alone (P = 0.028; F test). All error bars represent SEM.

Figure 2.

Concentration dependence of isoflurane peak I_Na inhibition. (A) Representative normalized I_Na time courses over a range of isoflurane (ISO) concentrations (indicated) for WT, G229A, and G219A. Individual I_Na responses obtained from a single cell exposed to ISO with initial control (CTL) and bracketing washout (WASH) are shown; other concentrations were obtained from individual cells. Responses are normalized to peak I_Na in CTL. Horizontal black lines indicate baseline. Inset shows voltage protocol (frequency, 0.167 Hz; V_h, holding potential). (B) Concentration-response relationships for peak I_Na inhibition for each channel. Peak I_Na in isoflurane (Peak I_ISO) was normalized by that of control (Peak I_CTL) and plotted versus isoflurane concentration (n = 3–10; mean ± SEM). Curves are logistic function fits with indicated IC₅₀ values, which were significantly different across all three channels (P < 0.0001; F test).

Figure 3.

Isoflurane effects on current inactivation. (A) A two-pulse voltage protocol was used to characterize the recovery time course from inactivation. Cells were held at the indicated holding potential (V_h), and two 500-ms test pulses to −10 mV were delivered separated by a range of recovery intervals at V_h (protocol delivery frequency, <0.1 Hz). Available fractional current (peak current amplitude of pulse 2/peak amplitude of pulse 1) was plotted against recovery interval for control (CTL) and isoflurane (ISO; n = 3–7; mean ± SEM). Curves are biexponential fits of recovery responses: WT (CTL, A_F = −0.57 ± 0.063, τ_F = 47 ± 6.3 ms, A_S = −0.41 ± 0.063, τ_S = 320 ± 60 ms, B = 0.98 ± 0.006; ISO, A_F = −0.8 ± 0.08, τ_F = 54 ± 7 ms, A_S = −0.21 ± 0.08, τ_S = 400 ± 200 ms, B = 1.0 ± 0.01); G229A (CTL, A_F = −0.29 ± 0.034, τ_F = 300 ± 56 ms, A_S = −0.14 ± 0.027, τ_S = 3,700 ± 2,400 ms, B = 0.98 ± 0.02; ISO, A_F = −0.53 ± 0.044, τ_F = 220 ± 29 ms, A_S = −0.25 ± 0.042, τ_S = 2,300 ± 720 ms, B = 0.99 ± 0.015); G219A (CTL and ISO, A_F = −0.4 ± 0.082, τ_F = 110 ± 28 ms, A_S = −0.58 ± 0.083, τ_S = 680 ± 105 ms, B = 0.98 ± 0.01). G229A ISO response is also shown scaled to the same amplitude as CTL. Inset is a bar graph showing the amplitude and time constants from the WT biexponential fit for control (white bars) and 0.8 mM isoflurane (red bars) with differences by paired Student’s t test: *, P < 0.05 (n = 7). Error bars are SEM. (B) A three-pulse voltage protocol characterized the onset time course of current inactivation. Cells were held at the indicated V_h followed by pulse 1 (−10 mV, 500 ms), a variable duration prepulse (V_pre), pulse 2 (−10 mV, 500 ms), and after at least 10 s at V_h, pulse 3 (−10 mV, 500 ms) to confirm preparation stability. Protocol deliveries were separated by >10 s. Available fractional current was plotted against prepulse duration for CTL and ISO (n = 3–6; mean ± SEM). Smooth curves are monoexponential fits (time constant, τ) of onset responses (WT, τ_ISO = 16 ± 30 s; G229A, τ_ISO = 60 ± 46 s; G219A, τ_ISO = 12 ± 46 s). (C) SSI relationships were characterized using a variation of the three-pulse voltage protocol used in B, in which the prepulse duration was 90 s throughout; protocol deliveries were separated by >10 s. Normalized SSI curves were obtained by plotting available fractional current against prepulse potential for CTL and 0.8 mM ISO (n = 3–11; mean ± SEM). Curves are Boltzmann function fits to the averaged data with associated parameters (V₅₀, voltage at half amplitude; slope factor: WT, V_50(CTL) = −72.6 ± 1.3 mV, V_50(ISO) = −89.3 ± 1.3 mV, slope_CTL = −6.4 ± 0.8 mV/e, slope_ISO = −6.0 ± 1.1 mV/e; G219A, V_50(CTL) = −96.6 ± 1.4 mV, V_50(ISO) = −122.1 ± 0.8 mV, slope_CTL = −6.5 ± 0.8 mV/e, slope_ISO = −4.6 ± 1.0 mV/e). V₅₀ values were significantly different between CTL and ISO fits for both WT and G219A (P < 0.0001; F test), whereas slope factors were not different.

Figure 4.

Kinetic analysis of current time courses. (A, top) Representative currents triggered by depolarization (to −10 mV using same protocol as in Fig. 1 A) for control (CTL) and 0.8 mM isoflurane (ISO) for the indicated channels. Current responses are plotted as the additive inverse. Current time courses are well described by fits of the biexponential function (dashed blue lines). In the following equations, the slower exponential is falling and describes inactivation (τ_inact), and the faster exponential is rising and describes activation (τ_act). Fitted parameters (time constant in ms): WT, I_CTL= 2,800 pA•[exp(−(t − 0.87)/126) − exp(−(t − 0.87)/4)], I_ISO = 3,300 pA•[exp(−(t − 0.39)/27) − exp(−(t − 0.39)/2.8)]; G229A, I_CTL= 1,570 pA•[exp(−(t − 0.86)/510) − exp(−(t − 0.86)/6.4)], I_ISO = 1,690 pA•[exp(−(t − 0.55)/183) − exp(−(t − 0.55)/4.6)]; G219A, I_CTL= 2,400 pA•[exp(−(t − 0.57)/20) − exp(−(t − 0.57)/3.4)], I_ISO = 1,750 pA•[exp(−(t − 0.27)/7) − exp(−(t − 0.27)/1.9); see Materials and methods. (bottom) Plots of fit residuals. (B) Responses from A replotted on an expanded timescale to focus on the early activation phase. The preponderance of the early time course is well described by the biexponential functions (dashed blue lines), except for the initial activation phase (marked by the arrow), which was not considered during biexponential function fitting; see Materials and methods. Insets show same data as in main figure plotted on an expanded timescale to show initial phase of activation time course. (C) Group time constants for inactivation (τ_inact) and activation (τ_act) phases for CTL and ISO, as indicated (n = 3–5; paired t test: *, P < 0.05; ***, P < 0.001). Control inactivation and activation time constants for G229A and G219A were also compared with WT (unpaired Student’s t test: ns, not significant; ††, P < 0.01; †††, P < 0.001). Error bars are SEM.

Figure 5.

Fitting of NaChBac current responses using the six-state Markov model. (A) A sequential six-state Markov model proposed by Kuzmenkin et al. (2004) to account for NaChBac gating based on ionic and gating current results. Four closed states (C_n) are visited as governed by forward (α₁) and backward (β₁) rate constants before the open state (O). O transitions to the inactivated state (I) governed by forward (α₂) and backward (β₂) rate constants. (B) The six-state model was used to analyze isoflurane microscopic gating effects manifest in families of mean (±SEM; n = 4–6) normalized P_o responses (empirical, coarse black and red lines) over a range of triggering voltages (−40 to 0 mV, voltage protocol shown in top, left, bottom inset) in control (top) and 0.8 mM isoflurane (bottom) for each channel as indicated. The fitted six-state model P_o responses (simulated, smooth gray and black lines) reproduced mean normalized P_o responses, including the activation phase (top insets), where “goodness of fit” is supported by residual plots (bottom, time bars 200 ms, 200 ms, and 100 ms for WT, G229A, and G219A, respectively). Optimal model parameter sets were estimated using the entire voltage family of mean normalized P_o responses as targets (see Materials and methods). Top insets show responses replotted on an expanded timescale to focus on the early activation time course. Below are serial presentations of associated fit residual plots at the indicated voltages.

Figure 6.

Isoflurane effects on NaChBac gating parameters analyzed using the six-state Markov model. Plots of estimated gating parameters with 95% confidence intervals for k_x(0) of rate constants (left) and z_x and x_x (right). Parameter estimation involved fitting model P_o responses to one family of mean normalized P_o responses for each experimental condition (see Fig. 5). 95% confidence intervals were determined for the final value of each estimated parameter. Negative confidence intervals were limited to zero. β₂ values were scaled up by 100 for clarity because values were uniformly less than one. Insets show estimated change in free energy of associated gating transition (ΔG_ISO) induced by isoflurane (see Materials and methods). Estimated values with 95% confidence interval of model parameters k_α1(0), k_β1(0), k_α2(0), k_β2(0) (s⁻¹) and z₁, x₁, z₂, x₂ (z_x[e0] and x_x [unitless]) are as follows: WT CTL: 978 ± 9.2, 45.1 ± 11.1, 9.33 ± 0.14, 0.19 ± 0.068, 1.94 ± 0.16, 0.32 ± 0.028, 0.26 ± 0.65, 0.74 ± 1.87; ISO: 1,450 ± 9.9, 36.1 ± 8.5, 33.1 ± 0.28, 0.11 ± 0.064, 1.86 ± 0.12, 0.29 ± 0.02, 0.067 ± 0.83, 0.45 ± 5.67; G229A CTL: 555 ± 5.13, 56.9 ± 8.9, 1.69 ± 0.048, 1.01 × 10⁻⁶ ± 0.084, 1.53 ± 0.10, 0.31 ± 0.025, 4.7 × 10⁻⁴ ± 2,700, 5.8 × 10⁻⁵ ± 354; ISO: 707 ± 5.25, 40.7 ± 7.7, 5.42 ± 0.075, 36.8 ± 3.8, 1.41 ± 0.094, 0.21 ± 0.017, 0.3 ± 0.15, 0.046 ± 0.088; G219A CTL: 1,190 ± 7.72, 38.5 ± 6.84, 40.3 ± 0.33, 8.77 ± 4.62, 1.84 ± 0.093, 0.37 ± 0.021, 0.22 ± 0.75, 0.44 ± 1.53; ISO: 1,740 ± 19.1, 80.4 ± 26.1, 133 ± 2.87, 0.039 ± 9.81, 1.4 ± 0.17, 0.39 ± 0.049, 4.2 × 10⁻⁶ ± 302, 6.7 × 10⁻⁵ ± 1,110, respectively. Estimated values with 95% confidence intervals of model scale factors K_−40mV, K_−30mV, K_−20mV, K_−10mV, and K_0mV are as follows: WT CTL: 0.91 ± 0.1, 0.86 ± 0.05, 0.94 ± 0.05, 1.12 ± 0.03, 1.18 ± 0.02; ISO: 0.82 ± 0.04, 0.98 ± 0.02, 1.09 ± 0.02, 1.17 ± 0.02, 1.18 ± 0.02; G229A CTL: 1.13 ± 0.14, 1.03 ± 0.06, 0.98 ± 0.04, 0.99 ± 0.02, 1.07 ± 0.02; ISO: 0.82 ± 0.04, 0.85 ± 0.02, 0.94 ± 0.02, 1.05 ± 0.02, 1.09 ± 0.02; G219A CTL: 0.91 ± 0.04, 1.03 ± 0.05, 1.07 ± 0.02, 1.13 ± 0.02, 1.17 ± 0.02; ISO: 0.87 ± 0.06, 0.85 ± 0.06, 0.88 ± 0.05, 0.937 ± 0.04, 0.93 ± 0.02, respectively.

(Scheme 2)