In vitro activity: Benzocaine blocks μ1 wild-type Na+ currents in a dose-dependent manner with IC50 of 0.8 mM in HEK293T cells. Benzocaine (1 mM) blocks about 55% of wild-type Na+ current but about 95% of μ1-N1584A mutant current. Benzocaine (1 mM) blocks about 55% of wild-type μ1 currents, but about 80% of μ1-I1575A mutant current. Benzocaine results in a biphasic (protective/inductive) concentration-dependent hemolytic effect upon rat erythrocytes, with an effective Benzocaine:lipid molar ratio in the membrane for protection (RePROT), onset of hemolysis (ReSAT) and 100% membrane solubilization (ReSOL) of 1.0:1, 1.1:1 and 1.3:1, respectively. Benzocaine and 4-hydroxybenzoate interact with the open and inactivated channels during repetitive pulses, but during the interpulse the complex dissociates too fast to accumulate sufficient use-dependent block of Na+ currents. Benzocaine (500 μM) reduces the peak and steady-state currents and increases the amplitude of the inactivating component from 21.7% to 30.2% (n=7, P<0.05), so that benzocaine-induced block at the end of pulses to +60 mV averaged 30.9% (n=7). Benzocaine (500 μM) significantly accelerates the initial phase of deactivation (τf=27.2±2.6 ms, n=7, P<0.01), but does not modify the slow phase of tail current decline. Benzocaine binds with high affinity to an intracellular binding site to produce 'agonist' effects and to a low affinity subsite, which is also located in the inner mouth, to produce the blocking effects. Benzocaine and extracellular K(+) interact to modify the voltage-dependence of channel opening.

Pflugers Arch. 1998 Jan;435(2):293-302; Biophys Chem. 2000 Oct 30;87(2-3):213-23; Biophys Chem. 2000 Oct 30;87(2-3):213-23.