Divergent effects of anesthetics on lipid bilayer properties and sodium channel function

Abstract

General anesthetics revolutionized medicine by allowing surgeons to perform more complex and much longer procedures. This widely used class of drugs is essential to patient care, yet their exact molecular mechanism(s) are incompletely understood. One early hypothesis over a century ago proposed that nonspecific interactions of anesthetics with the lipid bilayer lead to changes in neuronal function via effects on membrane properties. This model was supported by the Meyer-Overton correlation between anesthetic potency and lipid solubility and despite more recent evidence for specific protein targets, in particular ion-channels, lipid bilayer-mediated effects of anesthetics is still under debate. We therefore tested a wide range of chemically diverse general anesthetics on lipid bilayer properties using a sensitive and functional gramicidin-based assay. None of the tested anesthetics altered lipid bilayer properties at clinically relevant concentrations. Some anesthetics did affect the bilayer, though only at high supratherapeutic concentrations, which are unlikely relevant for clinical anesthesia. These results suggest that anesthetics directly interact with membrane proteins without altering lipid bilayer properties at clinically relevant concentrations. Voltage-gated Na⁺ channels are potential anesthetic targets and various isoforms are inhibited by a wide range of volatile anesthetics. They inhibit channel function by reducing peak Na⁺ current and shifting steady-state inactivation toward more hyperpolarized potentials. Recent advances in crystallography of prokaryotic Na⁺ channels, which are sensitive to volatile anesthetics, together with molecular dynamics simulations and electrophysiological studies will help identify potential anesthetic interaction sites within the channel protein itself.

Keywords: Amphiphiles; Anesthetic mechanisms; Bilayer modification; Gramicidin channel; Isoflurane; NaChBac.

Fig. 1

Schematic of gramicidin channels as molecular force probes for sensing changes in bulk lipid bilayer properties. Two non-conducting gramicidin monomers (green cylindrical structures) can dimerize to form a conducting dimer. The thickness of the bilayer (d₀) is larger than the length of the conducting dimer (l), therefore a bilayer deformation energy is required to enable this dimerization. Amphiphiles – compounds with both hydrophilic and lipophilic properties – can lead to changes in bulk bilayer properties that can be sensed by gramicidin channels.

Fig. 2

Schematic representation of potential drug-ion channel interaction sites. 1) binding within the pore impeding ion permeation, 2) drug binding sites formed by the channel, 3) specific drug binding sites composed of both the protein and the lipid bilayer, 4) drug accumulation at the protein-bilayer interface, 5) drug partitioning into the lipid bilayer-solution interface. Adapted from (Andersen 2008).

Fig. 3

Effects of isoflurane on bulk lipid bilayer properties using the gramicidin based fluorescence assay. Example of normalized fluorescence traces over a 200 ms time course showing three concentrations of isoflurane (expressed as minimum alveolar concentration, MAC, defined as the concentration that prevents movement in response to a painful stimulus in 50% of subjects, comparable to EC₅₀) with ethanol (5% EtOH; ~0.86 M) as a positive control. Gray dots denote results from all experiments (>5 per condition) and solid colored lines denote the average of all experiments for the individual condition. Note the absence of fluorescence decay using vesicles without gramicidin (−gA) and increasing rates of fluorescence decay in gramicidin-containing vesicles (+gA) with increasing concentrations of isoflurane.

Fig. 4

Effects of general anesthetics on lipid bilayer properties using the gramicidin based fluorescence assay. Normalized fluorescence quench rates are displayed for ether anesthetics (red), alkane anesthetics (orange) and intravenous anesthetics (yellow) at the clinical concentration of 1 MAC (or 10 μM for intravenous anesthetics). The value for 5% ethanol (EtOH; ~0.86 M) is shown as a positive control. Flurothyl* and F6* are compounds that do not cause immobility (and therefore are classified as nonanesthetics) that were tested at concentrations predicted to produce anesthesia based on their lipid solubilities. None of the tested anesthetics or nonanesthetics altered bulk lipid bilayer properties as detected by their normalized quench rates (Rate_drug/Rate_control), with a value of 1.0 indicating no significant effect on bulk lipid bilayer properties. Data are expressed as mean ± SD, n = 3–5. Adapted from (Herold et al. 2017).

Fig. 5

Isoflurane inhibition of eukaryotic and prokaryotic Na_v. a Macroscopic whole-cell Na⁺ current traces recorded from a mammalian ND7/23 cell endogenously expressing tetrodotoxin-sensitive Na_v in the absence (gray discontinued traces) or presence (purple traces) of 0.8 mM isoflurane (~2.5 MAC). From a holding potential (V_h) of −80 mV the alternating stimulation protocols were chosen to test voltage-dependent inhibition. A test pulse (10 ms at 0 mV) to elicit peak Na⁺ current was preceded by a 300-ms prepulse to either −130 mV (denoted “V₀”; left traces) or to a voltage at which approximately half of the channels were in the fast-inactivated state (denoted V_½; –70 mV; right traces). b Steady-state inactivation (or Na⁺ channel availability; h_∞) of tetrodotoxin-sensitive Na⁺ currents (Na_v1.4) was tested using a double-pulse protocol with a 30-ms prepulse of from −110 to −20 mV in 10-mV steps, followed by a 25-ms test pulse to −10 mV. Peak Na⁺ current was normalized (I_Na/I_Namax), plotted against prepulse potential, and fitted with a two-state Boltzmann distribution to calculate V_½, which was shifted by −10 mV in the presence of 0.8 mM isoflurane. Data are expressed as mean ± SD, n=7. Adapted from (Ouyang et al. 2009). c NaChBac current traces recorded from a transfected HEK293FT cell in the absence (gray discontinued traces) or presence (purple traces) of 0.8 mM isoflurane (~2.5 MAC) using the stimulation protocols depicted from a V_h of either −140 mV or −80 mV to test for voltage-dependent inhibition. The time scale over which NaChBac activates and inactivates is considerably slower than for mammalian Na_v (~500 ms vs. 2–3 ms, respectively); the accelerated NaChBac current decay in the presence of isoflurane is also noteworthy. d NaChBac steady-state inactivation was tested from a V_h of −140 mV with 90-s prepulses ranging from −140 to −40 mV followed by a test pulse to −10 mV. V_½ was shifted by −16 mV in the presence of 0.8 mM isoflurane. Data expressed as mean ± SD, n = 7–15. Adapted from (Sand et al. 2017).