Charged tetracaine as an inactivation enhancer in batrachotoxin-modified Na+ channels

Abstract

Two distinct types of local anesthetics (LAs) have previously been found to block batrachotoxin (BTX)-modified Na+ channels: type 1 LAs such as cocaine and bupivacaine interact preferentially with open channels, whereas type 2 LAs, such as benzocaine and tricaine, with inactivated channels. Herein, we describe our studies of a third type of LA, represented by tetracaine as a dual blocker that binds strongly with closed channels but also binds to a lesser extent with open channels when the membrane is depolarized. Enhanced inactivation of BTX-modified Na+ channels by tetracaine was determined by steady-state inactivation measurement and by the dose-response curve. The 50% inhibitory concentration (IC50) was estimated to be 5.2 microM at -70 mV, where steady-state inactivation was maximal, with a Hill coefficient of 0.98 suggesting that one tetracaine molecule binds with one inactivated channel. Tetracaine also interacted efficiently with Na+ channels when the membrane was depolarized; the IC50 was estimated to be 39.5 microM at +50 mV with a Hill coefficient of 0.94. Unexpectedly, charged tetracaine was found to be the primary active form in the blocking of inactivated channels. In addition, external Na+ ions appeared to antagonize the tetracaine block of inactivated channels. Consistent with these results, N-butyl tetracaine quaternary ammonium, a permanently charged tetracaine derivative, remained a strong inactivation enhancer. Another derivative of tetracaine, 2-(di-methylamino) ethyl benzoate, which lacked a 4-butylamino functional group on the phenyl ring, elicited block that was approximately 100-fold weaker than that of tetracaine. We surmise that 1) the binding site for inactivation enhancers is within the Na+ permeation pathway, 2) external Na+ ions antagonize the block of inactivation enhancers by electrostatic repulsion, 3) the 4-butylamino functional group on the phenyl ring is critical for block and for the enhancement of inactivation, and 4) there are probably overlapping binding sites for both inactivation enhancers and open-channel blockers within the Na+ pore.

Excitatory and inhibitory effects of tricaine (MS-222) on fictive breathing in isolated bullfrog brain stem

“…MS-222 blocks inward Na ϩ currents in the isolated squid axon (11), but the concentrations used in that study (1-3 mM) also abolish fictive breathing in the present study. Benzocaine and tricaine block voltage-gated Na ϩ channels in the inactivated state (44), which differs from the inhibitory effects of other local anesthetics, such as lidocaine and cocaine, which block voltage-gated Na ϩ channels in the open state (44). Voltage-gated Na ϩ channels from different tissues also exhibit varying sensitivities to local anesthetics with cardiac Na ϩ channels being more sensitive than nerve or skeletal muscle Na ϩ channels (25).…”

“…The most commonly used compound for anesthesia in fishes and amphibians is tricaine methanesulfonate (MS-222), a local anesthetic that is structurally similar to benzocaine, procaine, and lidocaine (19,29). Local anesthetics, such as benzocaine and MS-222, block action potential generation by altering the gating properties of voltage-gated Na ϩ channels (44). Although the efficacy of MS-222 as a general anesthetic for fishes and amphibians is well established (4,12,37,38), the mechanisms and sites of action for MS-222 remain unclear.…”

Inhibition of frog skeletal muscle sodium channels by newly synthesized chiral derivatives of mexiletine and tocainide

“…It has been long claimed that the charged fraction of a local anesthetic-like compound contributes to its use-dependent behaviour because it can gain access to the receptor in the sodium channel by permeating the hydrophilic pathway of the open channel from the cytoplasm (Schwarz et al 1977). In this way the charged moiety would be driven toward and away from the binding site not only in relation to the channel state, but also to the voltage gradient across the membrane (Wang et al 1993(Wang et al , 1994. In fact the presumed receptor for the local anesthetic-like compounds is on the S6 segment of the IV domain, nearby the inactivation gate of the III-IV cytoplasmic linker, located halfway the electric field and modification of the amino acid hydrophobicity in this region greatly modified the use-dependent block by lidocaine derivatives (Ragsdale et al 1994).…”

Reconsolidation of fresh, remote, and extinguished fear memory in medaka: old fears don't die

“…The Na + channel blocker MS222 (Wang et al ., 1994; EMEA, 1999) is an effective consolidation and reconsolidation blocker in medaka (Eisenberg et al ., 2003). It blocks long‐term memory when applied to the bath immediately after training but not afterwards, but has no detectable lingering effects on performance and short‐term memory after washout.…”