БИОЛОГИЧЕСКИЕ МЕМБРАНЫ, 2012, том 29, № 1-2, с. 13-20


УДК 577.352


© 2012 G.-K. Wang, G. R. Strichartz

Pain Research Center, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham & Women's Hospital

and Harvard Medical School, Boston MA 02115, USA; e-mail: gstrichz@zeus.bwh.harvard.edu Received 05.07.2011

Knowledge about the mechanism of impulse blockade by local anesthetics has evolved over the past four decades, from the realization that Na+ channels were inhibited to effect the impulse blockade to an identification of the amino acid residues within the Na+ channel that bind the local anesthetic molecule. Within this period appreciation has grown of the state-dependent nature of channel inhibition, with rapid binding and unbinding at relatively high affinity to the open state, and weaker binding to the closed resting state. Slow binding of high affinity for the inactivated state accounts for the salutary therapeutic as well as the toxic actions of diverse class I anti-arrhythmic agents, but may have little importance for impulse blockade, which requires concentrations high enough to block the resting state. At the molecular level, residues on the S6 transmembrane segments in three of the homologous domains of the channel appear to contribute to the binding of local anesthetics, with some contribution also from parts of the selectivity filter. Binding to the inactivated state, and perhaps the open state, involves some residues that are not identical to those that bind these drugs in the resting state, suggesting spatial flexibility in the "binding site". Questions remaining include the mechanism that links local anesthetic binding with the inhibition of gating charge movements, and the molecular nature of the theoretical "hydrophobic pathway" that may be critical for determining the recovery rates from blockade of closed channels, and thus account for both therapeutic and cardiotoxic actions.

Keywords: local anesthetics, sodium channel, drug binding, modulated receptor.


Although it had been known for more than 70 years that local anesthetics could produce numbness, and for about 30 years that this loss of sensation corresponded to a blockade of nerve impulses, the actual mechanism and site of action for this effect only emerged through modern electrophysiological and biochemical experiments. The first of these was the report of a strong, reversible inhibition of Na+ currents in the voltage-clamped squid giant axon by procaine [1]. Since the inward Na+ current was recognized as the quintessential feature of regenerative action potentials [2] this finding identified the major mode of action for impulse blockade by local anesthetics.

The squid axon continued to be a useful tool for studying the actions of local anesthetics, in a series of elegant experiments by Narahashi and colleagues in the 1970's, using the internal perfusion that, at that time, could only be accomplished with that large nerve structure. Internal perfusion allowed the direct application of the drug inside or outside the nerve's plasma membrane, and the control of the internal and external pH. In these experiments, Narahashi's team showed that the most potent action of tertiary amine local anesthetics occurred when the internally perfused drug was accompanied by a relatively acidic in-

ternal pH, suggesting that the cationic species acting from the intracellular side was most active in blocking the channel [3]. This conclusion was further supported by the observation that quaternary homologues of the smaller local anesthetics, such as the N-ethyl derivative of lidocaine, QX-314, were far more effective blockers when placed inside the axon rather than applied extracellularly [4]. Surprising later work, in frog muscle, showed that when a local anesthetic molecule was blocking the Na+ channel from the intercellular compartment, the protonation—deprotonation reaction of the tertiary amine (pK = 8—9 in solution) was dependent on the extracellular pH and virtually independent of the intracellular pH [5]! This observation, together with the control of the channel blocking and the unblocking rates by the open state of the channel [6—9] were consistent with a blocking site within the channel's pore. Some potency was still attributed to the neutral species however, based on the pH studies and on the findings that uncharged, non-ionizable derivatives of local anesthetics were able to inhibit Na+ currents [10—12].

Two seminal papers followed shortly after Nara-hashi's work, showing that the state transitions that underlay the gating (opening, inactivation) of Na+ channels were altered by local anesthetics [13] and

that such gating was itself an essential modulator of local anesthetic block [6]. Both observations lead to the proposition that the Na+ channel had a direct interaction with local anesthetic molecules, rather than being inhibited by some non-direct membrane perturbing activity [14]. Subsequent studies showed that the "gating currents", which resulted from the asymmetric movement of the charged regions of the Na+ channel that underlay and temporally preceded channel opening, were suppressed but not totally abolished by local anesthetics [15—18]. The channels, it thus appeared, were not simply inhibited by local anesthetic plugging of the ion-conducting pore, but rather were mechanically disrupted by the drug.

These observations formed the foundation for the Modulated Receptor Hypothesis, which viewed the interactions between channel and local anesthetic with the perspective of the dynamic binding of an enzymeinhibitor complex, with reciprocal interactions between allosteric transitions of the enzyme and state-dependent binding of the inhibitor.


As the preceding section has shown, block of Na+ currents by local anesthetics is most likely due to the existence of a local anesthetic receptor within the voltage-gated Na+ channels. The early framework regarding the local anesthetic receptor is best exemplified by the modulated receptor hypothesis put forward by Hille [19], initially proposed to explain the "use-dependent" block of sodium currents during repetitive pulses and the shift in apparent inactivation of the channel by local anesthetics [13, 20]. This hypothesis envisions that (1) the local anesthetic receptor site is modulated by state transitions during membrane depolarization and (2) the open and inactivated states of voltage-gated Na+ channels have higher affinities toward LA drugs than that of the resting state. Such a hypothesis implies that the receptor site is movable during state transitions, flexible in its receptor configuration, and perhaps participating in an induced-fit with its ligands upon depolarization. More specifically, as the Na+ channel cycles from resting to open and inactivated states during a pulse, the local anesthetic receptor site also changes its configuration accordingly, which in turn displays higher binding affinities toward LA drugs because of its conformational changes. It is plausible that inactivated states are "stabilized" by LAs through high-affinity binding and may be "locked" in such states during repetitive pulses. If the drug remains bound with the inactivated channels because of its slow dissociation from the LA receptor, more and more receptors will be occupied by LA drugs until the drug-bound and drug-free receptor reach a steady state during repetitive pulses. This modulated receptor hypothesis readily accounts for the use-dependent phenotype of LA drugs during repetitive pulses. It is now apparent that the implications of Hille's hypoth-

esis are far-reaching in the ion channel field. Numerous receptors for clinic drugs within ion channels are indeed modulated by ion channel gating.

The structure and function of S6 segments along with the LA receptor within voltage-gated Na+ channels. A

number of mammalian voltage-gated sodium channel proteins were delineated during 1980 and 1990 by molecular cloning techniques. The voltage-gated Na+ channel in excitable membranes consists of one large a-subunit and one or two smaller auxiliary p-subunits

[21]. To date, there are nine large a-subunit (Nav1.1— Nav1.9) and four p-subunit (p1—p4) isoforms found in mammals. The large a-subunit peptide alone can form a functional Na+ channel that carries Na+ currents when expressed in mammalian cells or in frog oocytes. Each a-subunit peptide contains four homologous domains (D1—D4), each with 6 transmembrane segments (S1—S6) (Fig. 1). The voltage sensor is located within S4 segment whereas the selectivity filter (DEKA locus) is situated within the S5—S6 linker (also known as the Pore region) in each of four homologous domains. The activation gate is probably located at the C-termini of the four S6 segments and the inac-tivation gate is likely formed by the IFM motif at the intracellular D3—D4 linker.

The structural aspects of the local anesthetic receptor have been inferred by site-directed mutagenesis so far. The receptor mapping of the local anesthetic binding site was first reported in 1994 by Catterall's group

[22]. Using an alanine scanning technique, these authors found that the middle of the D4—S6 segment contains two aromatic residues (phenylalanine, position 13 and tyrosine, position 20) that are critical for local anesthetic binding (Fig. 2). In particular, the LA binding affinity toward the inactivated state of the sodium channel was drastically reduced when each of these two residues is substituted with alanine. The mutation, however, has lesser effects on the resting affinity toward LAs. It is interesting to note that these two aromatic residues are conserved in all 9 a-subunit isoforms. Further detailed studies have found that the phenylalanine residue is the most important residue for LA binding. Substitution of a l

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