Sevoflurane inhibition of the developmentally expressed neuronal sodium channel Na_v1.3

Abstract

Neuronal voltage-gated sodium channels (Na_v) are major targets for the neurophysiological actions of general anesthetics. In the adult brain, cell type-specific effects on synaptic transmission are attributed to the differential sensitivity to volatile anesthetics of specific Na_v subtypes preferentially expressed in mature neurons (Na_v1.1, Na_v1.2, Na_v1.6). Comparatively, developing neurons are more excitable than mature neurons. We determined volatile anesthetic effects on Na⁺ currents mediated by Na_v1.3, the principal Na_v subtype expressed in developing neurons. Sevoflurane at clinical concentrations inhibited peak Na⁺ current of human Na_v1.3 heterologously expressed in HEK293T cells in a voltage-dependent manner, induced a − 6.1 mV hyperpolarizing shift in the voltage dependence of steady-state inactivation, and slowed recovery from fast inactivation. Na_v1.3-mediated Na⁺ currents also exhibited distinct activation properties associated with hyperexcitability, including prominent persistent currents and ramp currents, both of which were significantly reduced by sevoflurane. Na_v1.3 showed a more depolarized voltage dependence of steady-state inactivation than Na_v1.2, consistent with its higher propensity for sustained repetitive firing. Na_v1.2 exhibited minimal persistent and ramp currents, and these were unaffected by sevoflurane. These findings identify subtype-specific effects of sevoflurane on neuronal Na_v subtype electrophysiological properties, and suggest a mechanistic basis for increased anesthetic sensitivity and toxicity in early neuronal differentiation and maturation.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-15280-6.

Introduction

Voltage-gated Na⁺ channels (Na_v) regulate neuronal excitability^1–4 action potential (AP)-driven Ca²⁺ influx, and Ca²⁺-dependent neurotransmitter release^3,5–8 and are important targets for general anesthetics^9–12. Upon neuronal depolarization, Na_v rapidly activate then inactivate generating an AP, followed by a return to resting membrane potential¹³. Volatile anesthetics (VA) directly inhibit heterologously expressed Na_v¹¹ by reducing AP amplitude and nerve terminal depolarization in various neuronal preparations^14–17 including dissociated neurons from the hippocampus¹⁸ a major site of anesthetic actions¹⁹.

Four major Na_v subtypes (Na_v1.1, Na_v1.2, Na_v1.3, Na_v1.6) are expressed in the mammalian brain^20,21, with Na_v1.3 preferentially expressed during early development^22,23. In the hippocampus, variable expression of Na_v subtypes in specific neuronal populations influences cellular and network excitability due to differential presynaptic and postsynaptic localizations⁸. Mature inhibitory hippocampal interneurons express more Na_v1.1 compared to excitatory pyramidal neurons that express relatively more Na_v1.2 and Na_v1.6^24–26. These Na_v subtypes are inhibited by VAs in two ways: (1) stabilization of the inactivated state resulting in a shift of steady-state inactivation to more negative membrane potentials, reducing channel availability and slowing recovery from fast-inactivation, and (2) interaction with the open and/or resting state to produce tonic block^9,27,28. Voltage- and frequency-dependent block of Na_v^17,28,29 reduces AP amplitude^15,30 and neurotransmitter release^15,31 with Na_v subtype and neurotransmitter selectivity. For example, isoflurane decreases glutamate release more potently than GABA release from dissociated hippocampal neurons^32. This is likely mediated through greater inhibition of Na_v1.2 and Na_v1.6 in glutamatergic boutons compared with lesser inhibition of Na_v1.1 in GABAergic boutons^18,33.

Heterogeneity in Na_v expression also occurs during neuronal differentiation and development. Expression of Na_v subtypes with distinct activation properties renders developing neurons hyperexcitable compared to mature neurons²². Compared to other subtypes, Na_v1.3 recovers from inactivation four-fold faster and has slower closed-state inactivation, reducing the threshold for activation in neurons that express Na_v1.3, leading to sustained repetition firing^34–37 even with unchanged or absent stimuli. Slower inactivation or resurgent Na⁺ currents enhance repetitive firing and modulate overall neuronal excitability as opposed to AP initiation and propagation^38,39. Although persistent Na⁺ currents in acute hippocampal slices are inhibited by isoflurane^40, subtype-selective effects of anesthetic sensitivity on developmentally expressed Na_v have not been investigated. We examined the effects of sevoflurane on the function of Na_v1.3, a major neuronal subtype developmentally expressed in the immature mammalian brain. Comparative studies with Na_v1.2 provide a neurophysiological basis for developmental stage-specific impact on neuronal excitability.

Results

Effects of sevoflurane on peak Na⁺ current

The effects of sevoflurane on Na_v1.3 currents were tested using a periodic stimulation protocol that allowed us to determine voltage-dependent drug effects^10,41,42. To assess peak Na⁺ current (I_Na) inhibition, a depolarizing pulse to 0 mV was applied every 5 s, following a 100 ms prepulse to either V₀ (–120 mV), where most channels remained in an available resting state, or to a potential at which ~ 50% of channels were in an inactivated state V_½−inact (–47 ± 5 mV). Figure 1b shows the time course of I_Na inhibition during sevoflurane wash-in. The slow onset likely reflects drug partitioning into intracellular compartments rather than an intrinsically slow binding rate to the channel⁴³. Results are expressed as the ratio of peak I_Na in the presence of sevoflurane to control I_Na (Fig. 1a-c, Supplemental Table S1). Sevoflurane significantly inhibited peak I_Na following a prepulse to V_½−inact in a concentration-dependent manner. Time control (sham) experiments using external solution without sevoflurane showed no time-dependent inhibition (Supplemental Fig. S2a, b; Supplemental Table S3). In addition to reducing peak I_Na, sevoflurane also accelerated macroscopic current decay. For Na_v1.3, current decay of inactivation was best fit with a bi-exponential function, consistent with two components of inactivation, and sevoflurane significantly reduced the fast time constant τ_fast without affecting τ_slow (Supplemental Fig. S1a-c). In contrast, Na_v1.2 current decay was adequately fit with a mono-exponential function, and τ_fast was similarly reduced by sevoflurane (Supplemental Fig. S1d-f).

Fig. 1.

Inhibition of peak Na_v1.3 current by sevoflurane. Sevoflurane inhibited peak Na⁺ current (I_Na) in a voltage-dependent manner at concentrations equivalent to 0.5 MAC, 1 MAC, or 2 MAC (0.14 mM, 0.28 mM, or 0.57 mM). a, Macroscopic Na⁺ currents from a transfected HEK293T cell expressing Na_v1.3 were evoked using a periodic alternating stimulation protocol (see inset) in the absence (CTL, black traces) or presence (SEVO, orange traces) of 1 MAC sevoflurane (0.28 mM). Sevoflurane did not inhibit peak I_Na when the test pulse followed a prepulse to V₀, a potential at which all channels were in the resting state. However, sevoflurane significantly inhibited peak I_Na with a prepulse to V_½−inact, a potential at which half of the channels were in a fast-inactivated state. b, The time course of the effects of wash–in and wash–out of 1 MAC sevoflurane. c, Concentration-dependent inhibition of Na_v1.3 current by sevoflurane at 0.5, 1, and 2 MAC (for values, see Supplemental Table S1) following prepulse to V₀ or V_½−inact. All data are mean ± SD, n = 6–7, **P < 0.01 vs. control by paired two-tailed Student t-test (P = 0.0086 for 0.5 MAC; P = 0.0053 for 1 MAC; P = 0.0037 for 2 MAC).

Effects of sevoflurane on activation and inactivation

Sevoflurane caused a small but significant hyperpolarizing shift in the voltage dependence of channel activation, indicating that channels opened at slightly more negative potentials (Fig. 2a, b). This suggests that sevoflurane facilitates activation by lowering the voltage threshold for channel opening. Steady-state fast inactivation, also referred to as channel availability (or h_∞ from the Hodgkin-Huxley model), was tested using a double-pulse protocol (Fig. 2a, inset). Sevoflurane significantly shifted the voltage dependence of steady-state inactivation by − 6.1 ± 2.7 mV toward more hyperpolarized potentials (Fig. 2c, Supplemental Table S1). No such shift was seen in time-control (sham) experiments using external solution without sevoflurane, confirming no significant time-dependent shift in V_½−inact (Supplemental Fig. S2c, d; Supplemental Table S3). This indicates that a greater fraction of channels either transitioned into and/or remained in the inactivated state at any given potential, reducing the pool of available channels for subsequent activation. The shift in V_½−inact was concentration-dependent, with the largest effect observed at 2 MAC (Fig. 2d). These findings are consistent with sevoflurane stabilization of the inactivated state, thereby limiting Na_v availability, while also slightly modulating activation properties.

Fig. 2.

Effects of sevoflurane on Na_v1.3 activation and inactivation. a, Representative families of whole-cell inward I_Na evoked by depolarisation (inset) from a holding potential (V_h) of − 80 mV in the absence (CTL; left, black traces) or presence of sevoflurane (1 MAC; 0.28 mM) (SEVO; right, orange traces). b, c, Current-voltage relationship of channel activation displayed as normalized conductance (G/G_max), and of inactivation (I_Na/I_Namax). Sevoflurane shifted the voltage dependence of activation by − 6.2 ± 3.2 mV (V_½−activ−17.6 ± 6.0 mV for CTL [white circles] vs. −23.7 ± 6.0 mV for SEVO [orange circles], P = 0.0026, n = 7). Sevoflurane also shifted the voltage for half-maximal inactivation (V_½−inact) by − 6.1 ± 2.7 mV toward hyperpolarized potentials (V_½−inact −44.8 ± 7.0 mV for CTL [white circles] vs. −50.9 ± 7.3 mV for SEVO [orange circles], P < 0.0001, n = 11). d, This shift in V_½−inact was concentration-dependent, with the strongest effect of a − 10.4 ± 5.3 mV shift seen at 2 MAC sevoflurane. All data are mean ± SD; **P < 0.01, ****P < 0.0001 vs. control by paired two-tailed Student t-test.

Sevoflurane slows recovery from fast inactivation

We measured the effects of sevoflurane on recovery from inactivation using a holding potential of − 120 mV, at which the majority of channels were in a resting, closed state (Fig. 3a-c). Sevoflurane (1 MAC; 0.28 mM) significantly slowed the fast component of recovery, as reflected by an increase in the fast time constant τ_fast, while the slow component τ_slow was unaffected (Supplementary Table S1). This indicates that sevoflurane selectively impairs the rapid recovery of Na_v1.3 from fast inactivation, potentially prolonging the refractory period without altering slower recovery kinetics.

Fig. 3.

Effects of sevoflurane on Na_v1.3 recovery from fast inactivation. We used a two-pulse protocol from a holding potential (V_h) of − 120 mV, with two 10 ms test pulses to 0 mV separated by an inter-pulse interval of 1–200 ms (inset in (a) shows stimulation protocol). a, Overlaid macroscopic I_Na traces for the two pulses with increasing inter-pulse intervals in the absence (CTL, black traces) or presence (SEVO, orange traces) of sevoflurane (1 MAC; 0.28 mM). Peak I_Na of the second test pulse slowly recovers to control values with increasing inter-pulse durations. b, Data fitted to a bi-exponential equation and plotted against inter-pulse-interval in the absence (white circles, dotted line) or presence (orange circles, solid orange line) of sevoflurane. c, Recovery time constants τ_fast and τ_slow in the absence (white circles) or presence (orange circles) of sevoflurane. Sevoflurane increased τ_fast from 1.55 ± 0.4 ms to 2.07 ± 0.5 ms (P = 0.0073), thereby slowing recovery from fast inactivation; τ_slow was not affected with 55 ± 30 ms for CTL and 40 ± 30 ms for SEVO; P = 0.3789. Data are mean ± SD, n = 7, **P < 0.01 vs. control by paired two-tailed Student t-test.

Na_v1.3 and Na_v1.2 have distinct electrophysiological properties

We compared the effects of sevoflurane on Na_v1.3 and Na_v1.2 to investigate possible subtype-selective actions on developmentally distinct Na_v subtypes. Under control conditions, the voltage-dependent properties of Na_v1.3 were more depolarized compared to Na_v1.2, with the voltage for half-maximal activation (V_½−activ) and inactivation (V_½−inact) significantly shifted by + 10 mV (Figs. 2 and 4a-c, Supplemental Table S1,S2). These differences indicate that Na_v1.3 channels exhibit depolarized gating, enhancing their likelihood of opening during action potentials and maintaining availability over a wider voltage range, potentially influencing neuronal excitability.

Fig. 4.

Effects of sevoflurane on Na_v1.2 peak I_Na, steady-state fast inactivation, and recovery from fast inactivation. a, Representative families of whole-cell inward Na⁺ currents evoked by depolarization (inset) from a holding potential (V_h) of − 80 mV in the absence (CTL; left, black traces) or presence of sevoflurane (1 MAC; 0.28 mM) (SEVO; right, orange traces). b, c, Current-voltage relationship of channel activation displayed as normalized conductance (G/G_max) and inactivation (I_Na/I_Namax). Sevoflurane did not affect the voltage dependence of activation (V_½−activ −27.3 ± 5.0 mV for CTL [white diamonds] vs. −29.4 ± 4.2 mV for SEVO [orange diamonds], P = 0.0544, n = 6). However, sevoflurane shifted the voltage for half-maximal inactivation (V_½−inact) by − 6.1 ± 1.8 mV toward hyperpolarized potentials (V_½−inact −54.2 ± 5.0 mV for CTL [white diamonds] vs. −60.3 ± 6.2 mV for SEVO [orange diamonds], P < 0.0005, n = 6). d, Effect of sevoflurane on peak I_Na (stimulation protocol see Fig. 1a). Sevoflurane did not inhibit peak I_Na when the test pulse followed a prepulse to V₀, a potential at which all channels were in the resting state. However, with a prepulse to V_½−inact, a potential at which half of the channels were in the fast-inactivated state, sevoflurane inhibited peak I_Na (normalized peak I_Na 0.97 ± 0.05 for V₀, P = 0.2238 and 0.73 ± 0.08 for V_½−inact, P = 0.0005, n = 6). e, f, Recovery from fast inactivation was tested using the same protocol as in Fig. 3b. e, Recovery data (Pulse₂/Pulse₁) were fitted to a bi-exponential equation and plotted against inter-pulse-interval in the absence (white diamonds, dotted line) or presence (orange diamonds, solid orange line) of sevoflurane. f, Recovery time constants τ_fast and τ_slow in the absence (white diamonds) or presence (orange diamonds) of sevoflurane. Sevoflurane increased τ_fast from 1.7 ± 0.4 ms to 2.8 ± 0.7 ms (P = 0.0039), thereby slowing recovery from fast inactivation (τ_slow was not affected with 40 ± 30 ms for CTL and 70.2 ± 70 ms for SEVO; n = 6, P = 0.3257). See Supplemental Table S2 for all values. Data are mean ± SD, **P < 0.01, ***P < 0.001 vs. control by paired two-tailed Student t-test.

Sevoflurane at a clinically relevant concentration of 1 MAC (0.28 mM) produced similar effects on Na_v1.2 (Fig. 4a-f) as observed with Na_v1.3. Specifically, sevoflurane significantly inhibited peak I_Na of Na_v1.2 when the test pulse followed a prepulse to V_½−inact (− 56 ± 7 mV), a potential at which ~ 50% of channels are in a fast-inactivated state, but not with a prepulse to V₀ (− 120 mV) (Fig. 4d). The voltage dependence of steady-state fast inactivation was shifted by ~ − 6 mV toward more hyperpolarized potentials (Fig. 4b, c). Sevoflurane also slowed recovery from fast inactivation, with a significant increase in the fast time constant τ_fast (Fig. 4e, f). Sevoflurane did not significantly alter the activation properties of Na_v1.2, highlighting a more pronounced effect on Na_v1.3, where the drug modulates both activation and inactivation gating. This action might be particularly relevant in neurons where Na_v1.3 is upregulated, such as during development or following injury.

Effects of sevoflurane on persistent Na⁺ current

The distinct gating mechanisms of Na_v1.3 result in persistent Na⁺ current (I_NaP), which enhances repetitive firing and hyperexcitability during neuronal development^34,36. I_NaP allows subthreshold depolarizations to generate APs, but it is unclear whether and how this is modulated by VAs. A large I_NaP was generated by Na_v1.3 which was significantly inhibited by 1 MAC sevoflurane (Fig. 5a-c). I_NaP generated by Na_v1.3 was ~ 6-fold greater than for Na_v1.2, which was minimal and not further reduced by sevoflurane (–146 ± 161 pA for Na_v1.3 vs. − 23 ± 19 pA for Na_v1.2; Fig. 5d-f). To further validate this persistent current component of Na_v1.3, we conducted additional experiments using a prolonged depolarizing step (120 ms), which confirmed the presence of a sustained non-inactivating current throughout the pulse (Supplemental Fig. S3). This current was similarly reduced by sevoflurane, and full block by tetrodotoxin (TTX) confirmed that the residual current was not due to leak or non-specific conductance.

Fig. 5.

Effects of sevoflurane on Na_v1.3 and Na_v1.2 persistent Na⁺ current (I_NaP). I_NaP was tested using the depicted stimulation protocol (a, see inset). a, d, Overlaid averaged macroscopic Na⁺ currents in the absence (black traces) or presence (orange traces) of sevoflurane (1 MAC; 0.28 mM). Right panels show a zoomed view of the final 5 ms of the 20 ms depolarization, highlighting I_NaP (CTL black traces, SEVO orange traces, shaded areas represent SD). b, e, Absolute I_Na​P values in pA, calculated as the mean current of the final 5 ms. Sevoflurane reduced I_NaP in Na_v1.3 (from − 146 ± 161 pA for CTL [white circles] to − 65 ± 83 pA for SEVO [orange circles], P = 0.0144, n = 10) but had no effect on Na_v1.2 (from − 23 ± 19 pA for CTL [white diamonds] to − 25 ± 23 pA for SEVO [orange diamonds], n = 9). c, f, I_NaP expressed as a percentage of peak I_Na, to account for variability in cell size and peak I_Na​ amplitude across recordings. Sevoflurane reduced I_NaP in Na_v1.3 from 2.9 ± 1.2% [CTL, white circles] to 1.3 ± 0.9% [SEVO, orange circles], P < 0.0011, n = 9), but not in Na_v1.2 (from 1.0 ± 0.8% for CTL [white diamonds] to 0.7 ± 0.6% for SEVO [orange diamonds], n = 9). Data are mean ± SD, *P < 0.05, **P < 0.01 vs. control by paired two-tailed Student t-test.

Effects of sevoflurane on ramp currents

Inward Na⁺ currents during slow depolarizing ramps contribute to neuronal hyperexcitability by promoting repetitive firing. We examined the effects of sevoflurane on ramp-induced currents (I_NaR) using a depolarizing voltage ramp from − 120 mV to + 40 mV over 800 ms (0.2 mV/ms). I_NaR was quantified by integrating the inward I_Na over the voltage range from the activation threshold to the reversal potential, yielding the total charge transfer. To account for variability in channel expression levels among cells, this charge transfer was normalized to the peak I_Na elicited by a standard step depolarization, resulting in a normalized charge transfer value (pC/nA). Under control conditions, Na_v1.3 mediated a greater normalized charge transfer during the ramp protocol compared to Na_v1.2 (9.6 ± 2.0 pC/nA for Nav1.3 vs. 3.5 ± 1.0 pC/nA for Na_v1.2; data from Fig. 6b). Sevoflurane at a clinically relevant concentration of 1 MAC (0.28 mM) significantly reduced the normalized charge transfer associated with I_NaR in Na_v1.3-expressing cells (Fig. 6a, b). In contrast, sevoflurane did not alter the normalized charge transfer in Na_v1.2 (Fig. 6b), suggesting a subtype-specific effect on Na⁺ channel ramp currents.

Fig. 6.

Sevoflurane selectively inhibits ramp-evoked Na⁺ currents (I_NaR) in Na_v1.3 but not Na_v1.2. a, Mean of all ramp currents recorded in response to a slow depolarizing ramp from − 120 mV to + 40 mV over 800 ms (0.2 mV/ms) for Na_v1.3 (left panel) or Na_v1.2 (right panel). Top panels show stimulation protocol. Bottom panel shows mean ramp current trace in the absence (CTL, solid black trace) or presence (SEVO, solid orange trace) of sevoflurane (1 MAC; 0.28 mM). The shaded areas show SEM for control (CTL, grey area) or sevoflurane (SEVO, orange area). Currents are normalized to peak I_Na (evoked by a depolarizing step to 0 mV) and expressed as a percentage. b, Normalized charge transfer (pC/nA) was calculated by integrating inward I_Na from the voltage at which currents activate to the voltage at which currents reverse polarity, followed by normalization to peak I_Na. Under control conditions, Na_v1.3 showed greater normalized charge transfer than Na_v1.2 (9.6 ± 5.7 pC/nA for Na_v1.3 vs. 3.5 ± 2.7 pC/nA for Na_v1.2, P = 0.0226 by unpaired two-tailed Student t-test). Sevoflurane reduced charge transfer in Na_v1.3 (9.6 ± 5.7 to 5.6 ± 3.1 pC/nA, P = 0.0203, n = 8), but not in Na_v1.2 (3.5 ± 2.7 to 1.7 ± 1.3 pC/nA, P = 0.1680 n = 7). Data are presented as mean ± SD. *P < 0.05 vs. control by paired two-tailed Student t-test.

Discussion

The developmentally expressed Na⁺ channel Na_v1.3 is sensitive to modulation by the volatile anesthetic sevoflurane. While the effects of volatile anesthetics on Na_v1.2 have been characterized previously, their impact on developmentally expressed Na_v1.3 were previously unknown. We compared the effects of sevoflurane on the channel gating properties of human Na_v1.3 and Na_v1.2, the predominant Na⁺ channel subtypes expressed in immature and mature neurons, respectively. Under control conditions, Na_v1.3 exhibited gating at more depolarized potentials compared to Na_v1.2: both the voltage of half-maximal activation (V_½−activ) and inactivation (V_½−inact) were ~ 10 mV more positive in Na_v1.3. Despite these differences, sevoflurane produced a comparable hyperpolarizing shift in steady-state fast inactivation for both subtypes. The degree of voltage-dependent inhibition of peak I_Na and the slowing of recovery from fast inactivation were also similar between Na_v1.3 and Na_v1.2, suggesting that baseline differences in gating do not substantially alter the overall pattern of modulation by sevoflurane.

Persistent Na⁺ current (I_NaP) can drive intrinsic neuronal hyperexcitability⁴⁴ and has been implicated in various physiological and pathological processes, including pacemaking, neuronal injury, chronic pain, and epilepsy. We observed large I_NaP in cells expressing Na_v1.3, consistent with enhanced excitability of immature neurons. Sevoflurane reduced I_NaP in Na_v1.3 expressing cells, while Na_v1.2 had much smaller persistent currents than Na_v1.3, and were not further reduced by sevoflurane.

Ramp currents (I_NaR), which can enhance responses to subthreshold depolarizations by reducing action potential firing threshold⁴⁵were more pronounced in Na_v1.3 expressing cells. This observation is consistent with studies indicating that mutations in Na_v1.3 linked to neuronal hyperexcitability increase ramp and persistent currents. Selective inhibition of these currents could mediate developmental stage-specific effects of sevoflurane and possibly other anesthetics on neuronal excitability.

Multiple volatile anesthetics have been shown to inhibit all neuronal Na_v subtypes, including Na_v1.1, Na_v1.2, and Na_v1.6, leading to reduced AP amplitude in mature neurons at clinically relevant concentrations^11,15,31,33. In addition to shifting the voltage dependence of steady-state fast inactivation and slowing channel recovery, our findings show that sevoflurane also reduces I_NaP and I_NaR in Na_v1.3-expressing cells, providing a basis for previous observations that volatile anesthetics suppress transient Na⁺ current (I_NaT) and I_NaP, thereby reducing hippocampal excitability⁴⁰. Thus, during brain maturation, multiple Na_v1.3 gating mechanisms are likely targeted by volatile anesthetics to inhibit Na_v-activated Ca²⁺-influx and neurotransmitter release that contribute to the neurophysiological actions of anesthetics.

Modulation of neuronal activity by general anesthetics during early development can lead to persistent alterations in neuronal survival and function^46,47. The neurotoxic effects of general anesthetics administered at critical periods early in development in the mammalian brain^48–50 are associated with structural and functional changes, including alterations in dendritic spine dynamics, dendritic arborization, and synaptic protein expression^48–50. General anesthetics have selective effects on neurotransmission by depressing excitatory and enhancing inhibitory synaptic transmission, which potentially alters the balance of excitotoxicity from glutamatergic transmission in immature neuronal networks^51,52. The relative differences in anesthetic effects on excitatory or inhibitory neurons vary between anesthetic agents with synaptic inhibition by modulation of GABA_A receptors essential for intravenous agents such as propofol, while reduction of glutamatergic transmission both presynaptically and postsynaptically contributes to the neurodepressant effects of volatile agents like sevoflurane^53–55. These distinctions influence early deleterious actions as administration of drugs that attenuate GABA_A receptor activity or potentiate AMPA signaling during emergence from neonatal general anesthesia can ameliorate neuronal circuit dysfunction and learning deficits under propofol and sevoflurane, respectively^46,47. As Na_v1.3 is predominantly expressed in excitatory neurons⁵⁶ and drives persistent excitability, our findings suggest a potential role for Na_v1.3 in the effects of volatile anesthetic exposure to alter activity of immature, excitatory brain networks. Further investigation is needed to understand compensatory actions on inhibitory networks as well as regulation of Na_v1.3 by intravenous agents.

In native neurons, Na⁺ channels are composed of a pore-forming α-subunit and one or more auxiliary β-subunits (β1-β4). Auxiliary subunits are critical in regulating channel function, cell surface expression, and downstream signaling. Thus, co-expression with β-subunit(s) more accurately mimics endogenous neuronal physiology, particularly in heterologous expression systems such as HEK293 cells. Out of the four β-subunits, Na_vβ1 is ubiquitously expressed in the CNS and its structure-function effects are well-characterized compared to other subunits. It has strong modulatory effects on channel properties including voltage dependence, channel kinetics, recovery, and surface expression^57–59. Specifically, β1 subunit accelerates both activation and inactivation of Na_v1.3 currents, shifts steady-state inactivation to more hyperpolarized potentials, and speeds up recovery of Na_v1.3 from inactivation^59,60. In addition, β1 can enhance membrane trafficking and aid stabilization of Na_v1.3 at the cell surface. These properties lead to sharper Na_v1.3 Na⁺ currents and supports higher firing frequencies in cells^59,60. Although in slightly reduced magnitude, co-expression of the β1 subunit similarly modulates Na_v1.2 compared with Na_v1.3^61,62 allowing for reproducible and reliable data collection.

Our baseline Na_v1.3 activation properties (V_½−activ​) including I_NaP are consistent with previous reports^45,63–65. However, our V_½−inact values were notably more depolarized at ∼–45 mV compared to ∼–70 mV in those studies. Additionally, we observed larger ramp currents and normalized charge transfer in Na_v1.3-expressing cells than those reported by Vanoye et al.^64,65. These differences might stem from alterations in holding potentials (V_h) (we chose a more physiological V_h for certain experiments) or differences in auxiliary subunit expression, as we co-expressed only the β1-subunit, whereas Vanoye et al. included both β1 and β2. The β2 subunit is known to modulate inactivation gating and reduce persistent current amplitude^60,66,67which might account for the more depolarized inactivation and enhanced ramp currents observed in our study.

Multiple binding sites have been proposed for volatile anesthetic interactions with Na_v^68–70; further studies are required to determine whether differences in anesthetic binding to specific sites contribute to these subtype-selective effects of sevoflurane. Recent structure-function studies in bacterial Na⁺ channels has revealed that sevoflurane can displace membrane lipids and bind within a conserved hydrophobic pocket formed between the S1–S4 helices of one subunit and the S5 helix of an adjacent subunit⁷¹. Whether a homologous site exists, or is functionally relevant, within the pseudotetrameric domains of mammalian Na_v remains an important question for future work. Various volatile anesthetics might exhibit different affinities for Na_v subtypes^72,73with sevoflurane showing stronger interactions with Na_v1.3 compared with Na_v1.2. Moreover, Na_v subtypes not only show cell-type specific expression⁷ but also distinct subcellular and cellular localizations⁸. Parvalbumin-expressing interneurons are enriched in Na_v1.1, while excitatory pyramidal neurons express more Na_v1.2, Na_v1.3, and Na_v1.6^7,56,74,75. While all Na_v subtypes are found at the axon initial segment (AIS) to some degree, Na_v1.1 and Na_v1.2 are preferentially expressed in the proximal AIS⁷⁶potentially allowing direct targeting of AP initiation by anesthetics.

The halogenated ethers isoflurane, sevoflurane, and desflurane represent the principal volatile anesthetics employed clinically in modern anesthesia. However, isoflurane and sevoflurane are more commonly used due to their safety profiles and cost-effectiveness. Considerable evidence from our group^{10,11,33,70,77–80} and others^81–84 show inhibition of neuronal Na_v by isoflurane with subtype-specificity. For example, Na_v1.1 is less sensitive to isoflurane compared to Na_v1.2 and 1.6 due to its distinct gating properties³³. Although similar, sevoflurane has distinct differences in pharmacological actions and clinical use, gaining broader acceptance and use in modern anesthesia practice. For instance, sevoflurane’s rapid onset and offset leads to faster emergence and recovery and causes less airway irritation than isoflurane^85,86. These characteristics make sevoflurane more suitable for various types of surgery, including in pediatric populations with increased Na_v1.3 expression⁸⁴. Although sensitivity to sevoflurane for neuronal Na_v has been reported^81,84,87, systematic effects of sevoflurane on selective neuronal Na_v subtypes or comparative effects between Na_v subtypes have not been reported. Here, we report agent-specific differences in efficacy for Na⁺ channel inhibition that could underlie differences in modulation of immature and mature brain networks.

Further studies of the selective effects of sevoflurane on Na_v subtypes could lead to the development of more selective and potentially safer anesthetic agents that selectively target specific Na_v subtypes based on neuronal stage or neuropathological state by reducing deleterious off target effects or specifically targeting desired effects.

Materials and methods

Materials

Sevoflurane was obtained from Henry Schein Medical (Melville, NY). HEK293T cells were from ATCC (Manassas, VA). All other chemicals were from Sigma-Aldrich (St. Louis, MO).

cDNA constructs

Human Na_v1.2-pIR-CMV-IRES-mScarlet (Accession number NM_021007) and human Na_v1.3-pIR (Accession number NM_001081676) cDNA were kindly provided by Professor A.L. George Jr. (Northwestern University, Evanston, IL) via AddGene (Watertown, MA) and Professor J. A. Kearney (Northwestern University, Evanston, IL), respectively.

Cell culture and electrophysiological recordings of Na⁺ currents

Human HEK293T cells (#CRL-3216, ATCC, Manassas, VA) were seeded into 35-mm dishes in Dulbecco’s Modified Eagle′s Medium (Sigma-Aldrich) supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 100 mg/ml streptomycin and incubated in a humidified atmosphere of 5% CO₂ and 95% air at 37 °C. HEK293T cells were cotransfected with cDNA for hNa_v1.2 or hNa_v1.3 (3–4 µg) and human auxiliary hβ1 subunit (0.8–1 µg) using Lipofectamine LTX (Invitrogen, Carlsbad, CA). Additionally, hNa_v1.3 was cotransfected with 0.8 µg pEGFP-N1 (Clontech, Mountain View, CA) as a reporter to allow identification of EGFP-transfected cells by fluorescence microscopy. Cells were re-seeded at lower density onto 12-mm glass coverslips 20–24 h post-transfection, and electrophysiological studies were conducted at least 3 h later to allow cells to adhere.

For electrophysiological recordings, coverslips were transferred into a small volume laminar-flow perfusion chamber (Warner Instruments, Hamden, CT) and continuously perfused at ~ 2 ml/min with extracellular solution containing (in mM): 130 mM NaCl, 10 mM HEPES, 3.25 mM KCl, 2 mM MgCl₂, 2 mM CaCl₂, 0.1 mM CdCl₂, and 20 mM TEA-Cl, adjusted to pH 7.4 (with NaOH) and to 310 mOsm/kg. Na⁺ currents were recorded in voltage-clamp mode at room temperature (23–24 °C) using an Axopatch 200B patch-clamp amplifier (Molecular Devices, San Jose, CA) with a 5 kHz low-pass filter at a sampling rate of 20 kHz. Recording pipettes were pulled from borosilicate glass capillaries (Sutter Instruments, Novato, CA) on a P-1000 horizontal puller (Sutter Instruments) and fire-polished before use. Pipette resistance was ~ 1.5–2.5 MΩ when filled with internal solution containing (in mM): 120 mM CsF, 10 mM NaCl, 10 mM HEPES, 10 mM EGTA, 10 mM TEA-Cl, 1 mM CaCl₂, and 1 mM MgCl₂ adjusted to pH 7.3 (with CsOH) and to 312 mOsm/kg (with sucrose). Access resistance (~ 2–4 MΩ) was reduced using 75–85% series resistance correction. To minimize space-clamp and series resistance errors, only cells expressing 1–8 nA peak current were analyzed. Initial whole cell seal resistance was 1–3 GΩ, and recordings were discarded if resistance dropped below 1 GΩ. Liquid–junction potentials were not corrected. Capacitive current transients were electronically canceled with the internal amplifier circuitry, and leak currents were digitally subtracted using the P/4 protocol⁸⁸ Recordings began 5 min after attaining the whole-cell patch configuration to allow equilibration of pipette solution and cytosol. Stimulation protocols were applied in control external solution, and again after a 3-minute superperfusion with external solution containing sevoflurane.

Sevoflurane superfusion

A saturated stock solution of sevoflurane in external solution (~ 5.2 mM at 23 °C) was diluted to the desired final concentration in a gas-tight glass syringe. Sevoflurane solutions were superfused using a pressure-driven microperfusion system (ALA Scientific, Westbury, NY) with a 200 μm diameter perfusion pipette tip positioned ~ 200 μm from the recorded cell. Sevoflurane concentrations sampled at the perfusion pipette tip were determined by gas chromatography using a Shimadzu GC-2010 Plus gas chromatograph (Shimadzu, Tokyo, Japan) after extraction into octane⁸⁹. An aqueous sevoflurane solution of 0.28 mM was used as the predicted minimum alveolar concentration (MAC; equivalent to the EC₅₀ for immobilization) in humans after temperature adjustment to 24°C^30,90. To ensure reproducibility and minimize variability, the time from break-in to initial control recordings was standardized across all experiments, as was the duration of sevoflurane exposure before drug measurements. This protocol was based on previously validated methods shown to maintain stable gating properties over time in similar experimental conditions^10,42,70. In addition, sham (time control) recordings were performed in a subset of cells that underwent identical timing and perfusion procedures using external solution without sevoflurane. These experiments confirmed the absence of time-dependent shifts in the voltage-dependence of inactivation or peak current amplitude (see Supplemental Fig. S2 and Supplemental Table S3), thereby supporting the specificity of drug-induced effects reported in the main text.

Stimulation protocols and data analysis

The holding potential (V_h) was − 80 mV unless otherwise stated. Voltage-dependent inhibition of peak inward Na⁺ current (I_Na) was analyzed using a 20 ms test pulse to 0 mV preceded by a 100 ms prepulse alternating between either − 120 mV (V₀) or the voltage of half-maximal inactivation (V_½−inact = − 47 ± 5 mV) applied every 5 s^10,41,42. V_½−inact was determined for each individual cell using the double-pulse protocol for steady-state inactivation described below. For voltage-dependence of activation, conductance (G) values were derived from the current-voltage (I-V) relationship using:

where I is the peak Na⁺ current (I_Na) at a given command voltage (V), and V_rev ​ is the measured Na⁺ reversal potential. Conductance values were normalized to maximal conductance (G/G_max), and the activation curve was fitted using a Boltzmann function:

where V_½-act is the voltage at which activation is half-maximal, and k is the slope factor describing the voltage sensitivity of activation.

Steady-state fast inactivation (h_∞), was measured using a double-pulse protocol with a 100 ms prepulse ranging from − 110 to + 30 mV in 10 mV steps, followed by a 20 ms test pulse to 0 mV. Peak currents during the test pulse were normalized to the maximal current (I_Na/I_Namax, where I_Namax is the maximal current that is elicited at the test potential), plotted against the prepulse potential (V_m), and fitted with a Boltzmann function:

where z_1/2 and V_½−inact denote the apparent gating valence and potential for half-maximal inactivation, respectively.

Recovery from fast inactivation was tested from a holding potential of − 130 mV using a two-pulse protocol with two identical 10 ms pulses to 0 mV separated by an inter-pulse interval with increasing durations from 1 to 200 ms. Current amplitudes were normalized (Pulse₂/Pulse₁), plotted against inter-pulse interval. The recovery time course was best fitted with a bi-exponential equation to obtain the recovery time constants τ:

where Y_t denotes the normalized current at time t, Y₀ denotes the normalized current at zero time, A_fast and A_slow represent the amplitudes of the fast and slow components, t is the inter-pulse interval, and τ_fast and τ_slow denote the fast and slow recovery time constants, respectively.

Statistical significance was assessed either by one-way ANOVA or by two-tailed paired Student t test. P < 0.05 was considered statistically significant. Programs used for data acquisition and analysis were pClamp 10 (Molecular Devices), Excel (Microsoft, Redmond, WA), and Prism 10 (GraphPad Software Inc., La Jolla, CA). Values are reported as mean ± SD unless otherwise stated.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

Na_v

Voltage-gated sodium channel

SEVO

Sevoflurane

VA

Volatile anesthetic

AP

Action potential

TEA-Cl

Tetraethylammonium chloride

Author contributions

KFH, HCH, and JP contributed to study conception and design. Material preparation, data collection, and analysis were performed by JX and KFH. Figures were prepared by KFH. The first draft of the manuscript was written by KH and JP and all authors reviewed previous versions of the the manuscript. All authors approved the final manuscript.

Funding

US National Institutes of Health grant R01 GM58055 to HCH, and R01 GM130722 to JP.

Data availability

The datasets generated and analyzed during the current study are included in this published article and its supplementary information files.

Declarations

Competing interests

The authors declare no competing interests.