CHARGED TETRACAINE AS AN INACTIVATION ENHANCER IN BATRACHOTOXIN-MODIFIED NA+ CHANNELS

Two distinct types of local anesthetics (LAs) have previously been fou nd 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 inactivate d channels. Herein, we describe our studies of a third type of LA, rep resented by tetracaine as a dual blocker that binds strongly with clos ed 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 me asurement and by the dose-response curve. The 50% inhibitory concentra tion (IC50) was estimated to be 5.2 mu M 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. Tetra caine also interacted efficiently with Na+ channels when the membrane was depolarized; the IC50 was estimated to be 39.5 mu M at +50 mV with a Hill coefficient of 0.94. Unexpectedly, charged tetracaine was foun d to be the primary active form in the blocking of inactivated channel s. In addition, external Na+ ions appeared to antagonize the tetracain e block of inactivated channels. Consistent with these results, N-buty l tetracaine quaternary ammonium, a permanently charged tetracaine der ivative, remained a strong inactivation enhancer. Another derivative o f tetracaine, 2-(dimethylamino) ethyl benzoate, which lacked a 4-butyl amino functional group on the phenyl ring, elicited block that was sim ilar to 100-fold weaker than that of tetracaine. We surmise that 1) th e binding site for inactivation enhancers is within the Na+ permeation pathway, 2) external Na+ ions antagonize the block of inactivation en hancers by electrostatic repulsion, 3) the 4-butylamino functional gro up 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 Napore.