Hille (1977. Journal of General Physiology. 69:497-515) first proposed a modulated receptor hypothesis (MRH) to explain the action of benzocaine in voltage-gated Na+ channels. Using the MRH as a framework, we examined benzocaine binding in batrachotoxin (BTX)-modified Na+ channels under voltage-clamp conditions using either step or ramp command signals. We found that benzocaine binding is strongly voltage dependent. At -70 mV, the concentration of benzocaine that inhibits 50% of BTX-modified Na+ currents in GH3 cells (IC50) is 0.2 mM, whereas at +50 mV, the IC50 is 1.3 mM. Dose-response curves indicate that only one molecule of benzocaine is required to bind with one BTX-modified Na+ channel at -70 mV, whereas approximately two molecules are needed at +50 mV. Upon treatment with the inactivation modifier chloramine-T, the binding affinity of benzocaine is reduced significantly at -70 mV, probably as a result of the removal of the inactivated state of BTX-modified Na+ channels. The same treatment, however, enhances the binding affinity of cocaine near this voltage. External Na+ ions appear to have little effect on benzocaine binding, although they do affect cocaine binding. We conclude that two mechanisms underlie the action of local anesthetics in BTX-modified Na+ channels. Unlike open-channel blockers such as cocaine and bupivacaine, neutral benzocaine binds preferentially with BTX-modified Na+ channels in a closed state. Furthermore, benzocaine can be modified chemically so that it behaves like an open-channel blocker. This compound also elicits a use-dependent block in unmodified Na+ channels after repetitive depolarizations, whereas benzocaine does not. The implications of these findings for the MRH theory will be discussed.

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