Daniel S. Duch

Central Nervous System Sodium Channels Are Significantly Suppressed at Clinical Concentrations of Volatile Anesthetics

Background Although voltage-dependent sodium channels have been proposed as possible molecular sites of anesthetic action, they generally are considered too insensitive to be likely molecular targets. However, most previous molecular studies have used peripheral sodium channels as models. To examine the interactions of volatile anesthetics with mammalian central nervous system voltage-gated sodium channels, rat brain IIA sodium channels were expressed in a stably transfected Chinese hamster ovary cell line, and their modification by volatile anesthetics was examined. Methods Sodium currents were measured using whole cell patch clamp recordings. Test solutions were equilibrated with the test anesthetics and perfused externally on the cells. Anesthetic concentrations in the perfusion solution were determined by gas chromatography. Results All anesthetics significantly suppressed sodium currents at clinical concentrations. This suppression occurred through at least two mechanisms: (1) a potential-independent suppression of resting or open sodium channels, and (2) a hyperpolarizing shift in the voltage-dependence of channel inactivation resulting in a potential-dependent suppression of sodium currents. The voltage-dependent interaction results in IC50 values for anesthetic suppression of sodium channels that are close to clinical concentrations at potentials near the resting membrane potential. Conclusions Contrary to the hypothesis that sodium channels are insensitive to general anesthetics, the results presented here indicate that current through central nervous system sodium channels examined at physiologic membrane potentials is significantly blocked by clinical concentrations of volatile anesthetics. This anesthetic interaction with sodium channels is voltage-dependent, consistent with a state-dependent modulated receptor model in which anesthetics more strongly affect the inactive state of the channel than the resting state.

Suppression of Central Nervous System Sodium Channels by Propofol

BACKGROUND Previous studies have provided evidence that clinical levels of propofol alter the functions of voltage-dependent sodium channels, thereby inhibiting synaptic release of glutamate. However, most of these experiments were conducted in the presence of sodium-channel activators, which alter channel inactivation. This study electrophysiologically characterized the interactions of propofol with unmodified sodium channels. METHODS Sodium currents were measured using whole-cell patch-clamp recordings of rat brain IIa sodium channels expressed in a stably transfected Chinese hamster ovary cell line. Standard electrophysiologic protocols were used to record sodium currents in the presence or absence of externally applied propofol. RESULTS Propofol, at concentrations achieved clinically in the brain, significantly altered sodium channel currents by two mechanisms: a voltage-independent block of peak currents and a concentration-dependent shift in steady-state inactivation to hyperpolarized potentials, leading to a voltage dependence of current suppression. The two effects combined to give an apparent concentration yielding a half-maximal inhibitory effect of 10 microM near the threshold potential of action potential firing (about -60 mV). Propofol inhibition was also use-dependent, causing a further block of sodium currents at these anesthetic concentrations. CONCLUSIONS In these experiments with pharmacologically unaltered sodium channels, propofol inhibition of currents occurred at concentrations about eight-fold above clinical plasma levels and thus at brain concentrations reached during clinical anesthesia. Therefore, the results indicate a possible role for sodium-channel suppression in propofol anesthesia.

Differential effects of anesthetic and nonanesthetic cyclobutanes on neuronal voltage-gated sodium channels

Background Despite their key role in the generation and propagation of action potentials in excitable cells, voltage-gated sodium (Na+) channels have been considered to be insensitive to general anesthetics. The authors tested the sensitivity of neuronal Na+ channels to structurally similar anesthetic (1-chloro-1,2,2-trifluorocyclobutane; F3) and nonanesthetic (1,2-dichlorohexafluorocyclobutane; F6) polyhalogenated cyclobutanes by neurochemical and electrophysiologic methods. Methods Synaptosomes (pinched-off nerve terminals) from adult rat cerebral cortex were used to determine the effects of F3 and F6 on 4-aminopyridine– or veratridine-evoked (Na+ channel–dependent) glutamate release (using an enzyme-coupled spectrofluorimetric assay) and increases in intracellular Ca2+ ([Ca2+]i) (using ion-specific spectrofluorimetry). Effects of F3 and F6 on Na+ currents were evaluated directly in rat lumbar dorsal root ganglion neurons by whole-cell patch-clamp recording. Results F3 inhibited glutamate release evoked by 4-aminopyridine (inhibitory concentration of 50% [IC50] = 0.77 mM [∼ 0.8 minimum alveolar concentration (MAC)] or veratridine (IC50 = 0.42 mM [∼ 0.4 MAC]), and veratridine-evoked increases in [Ca2+]i (IC50= 0.5 mM [∼ 0.5 MAC]) in synaptosomes; F6 had no significant effects up to 0.05 mM (approximately twice the predicted MAC). F3 caused reversible membrane potential–independent inhibition of peak Na+ currents (70 ± 9% block at 0.6 mM [∼ 0.6 MAC]), and a hyperpolarizing shift in the voltage-dependence of steady state inactivation in dorsal root ganglion neurons (−21 ± 9.3 mV at 0.6 mM). F6 inhibited peak Na+ currents to a lesser extent (16 ± 2% block at 0.018 mM [predicted MAC]) and had minimal effects on steady state inactivation. Conclusions The anesthetic cyclobutane F3 significantly inhibited Na+ channel–mediated glutamate release and increases in [Ca2+]i. In contrast, the nonanesthetic cyclobutane F6 had no significant effects at predicted anesthetic concentrations. F3 inhibited dorsal root ganglion neuron Na+ channels with a potency and by mechanisms similar to those of conventional volatile anesthetics; F6 was less effective and did not produce voltage-dependent block. This concordance between anesthetic activity and Na+ channel inhibition supports a role for presynaptic Na+ channels as targets for general anesthetic effects and suggests that shifting the voltage-dependence of Na+ channel inactivation is an important property of volatile anesthetic compounds.

The Sodium Channel from Electrophorus electricusa

The purification of the sodium channel from excitable tissues has been a crucial step toward the elucidation of channel mechanism and molecular biology. In turn, this advance required the development of assays for channels during their fractionation from disrupted and solubilized membranes (which obviously could not be based on their normal physiological role as voltage-gated mediators of ion transport into intact cells). Fortunately, such precise and sensitive assays have been achieved through the channel-specific, high affinity binding of tetrodotoxin (TTX) and saxitoxin.”’ In addition, when bound to the channel these toxins prevent their own binding site from denaturing during the lengthy purification procedures employed, allowing a high proportion of channel protein to be purified which retains the ability to bind toxin.3 Thus both TTX and saxitoxin have played a vital role in the study of the molecular biology of the sodium channel. Naturally successful purification of sodium channels also requires a rich tissue source which is readily available and amenable to biochemical isolation techniques. To the naive reader the use of the electric eel Electrophorus electricus as a source of channel material may seem unnecessarily exotic, but in fact these beasts are endowed with amazingly large quantities of sodium channels a t high d e n ~ i t y . ~ As a result, the eel electroplax channel has played a prominent role in channel studies, having now been p~rified,’.~ chemically characterized5 and sequenced,6 and visualized both by electron microscopy’ and immunocytochemical techniques? In addition, the function and pharmacology of the eel channel appear to be very similar to channels from other animals, so that there is reason to suppose that molecular information derived from electroplax channels will be broadly applicable to those from other sources. The success of the electric organ preparation is probably due as much to the richness of the tissue in sodium channels as to the skill (and luck!) of the investigators. A crude fraction of electroplax membranes has about 0.5% sodium channel relative to

The Membrane Lipid Cholesterol Modulates Anesthetic Actions on a Human Brain Ion Channel

Background Molecular theories of general anesthesia often are divided into two categories: (l) Anesthetics may bind specifically to proteins, such as ionic channels, and alter their function directly, and (2) anesthetics may alter the functions of integral membrane proteins indirectly through modification of the physical properties of the membrane. Recent studies have provided evidence that anesthetics can bind to proteins and modify their function directly, bringing into question the role of the membrane in anesthetic interactions. To reexamine the role of membrane lipids in anesthetic interactions, an experimental approach was used in which the membrane lipid composition could be systematically altered and the impact on anesthetic interactions with potential targets examined. Methods Sodium channels from human brain cortex were incorporated into planar lipid bilayers with increasing cholesterol content. The anesthetic suppression of these channels by pentobarbital was quantitatively examined by single channel measurements under voltage‐clamp conditions. Results Changes in cholesterol content had no effect on measured channel properties in the absence of anesthetic. In the presence of pentobarbital, however, cholesterol inhibited anesthetic suppression of channel ionic currents, with 1.9% (weight/weight, corresponding to 3.5 mol%) cholesterol decreasing anesthetic suppression of sodium channels by half. Conclusions These results support a critical role for the lipid membrane in some anesthetic actions and further indicate that differences in lipid composition must be considered in the interpretation of results when comparing the anesthetic potencies of potential targets in model systems.

Neurotoxin-modulated uptake of sodium by highly purified preparations of the electroplax tetrodotoxin-binding glycopeptide reconstituted into lipid vesicles

SummaryUsing the dialysable detergent CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate), the tetrodotoxin-binding protein from the electroplax of the electric eel has been purified to a high degree of both chemical homogeneity and toxin-binding activity. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the best preparations showed only a single microheterogeneous band atMr approximately 260,000, despite attempts to visualize smaller bands by sample overloading. Upon dialysis, this material became incorporated into the membranes of small unilamellar vesicles, and in this form the purified protein exhibited tetrodotoxin-binding properties similar to the component in the original electroplax membrane. Furthermore, in the presence of activator neurotoxins the vesicles were able to accumulate isotopic sodium in a manner similar to that previously described for less active or less pure preparations of vesicles containing either mammalian or eel electroplax toxinbinding proteins. Quantitative consideration of the isotopic transport activity of this pure material, along with the high degree of purity of the protein, strongly suggests that the 260-kDa glycopeptide from electroplax is necessary and sufficient to account for the sodium channel function seen in these studies, and eliminates the possible involvement of smaller peptides in the channel phenomena observed.

Spontaneous opening at zero membrane potential of sodium channels from eel electroplax reconstituted into lipid vesicles

SummaryThe voltage-dependent sodium channel from the eel electroplax was purified and reconstituted into vesicles of varying lipid composition. Isotopic sodium uptake experiments were conducted with vesicles at zero membrane potential, using veratridine to activate channels and tetrodotoxin to block them. Under these conditions, channel-dependent uptake of isotopic sodium by the vesicles was observed, demonstrating that a certain fraction of the reconstituted protein was capable of mediating ion fluxes. In addition, vesicles untreated with veratridine showed significant background uptake of sodium; a considerable proportion of this flux was blocked by tetrodotoxin. Thus these measurements showed that a significant subpopulation of channels was present that could mediate ionic fluxes in the absence of activating toxins. The proportion of channels exhibiting this behavior was dependent on the lipid composition of the vesicles and the temperature at which the uptake was measured; furthermore, the effect of temperature was reversible. However, the phenomenon was not affected by the degree of purification of the protein used for reconstitution, and channels in resealed electroplax membrane fragments or reconstituted, solely into native eel lipids did not show this behavior. The kinetics of vesicular uptake through these spontaneously-opening channels was slow, and we attribute this behavior to a modification of sodium channel inactivation.