OBJECTIVES: It has been demonstrated that at nanomolar concentrations benzocaine increased, whereas at micromolar concentrations, it blocked hKv1.5 channels in a voltage-dependent manner and modified the voltage-dependence of channel activation. The present study was undertaken to localize the putative binding sites involved in the 'agonists' and blocking effects of benzocaine. METHODS: Experiments were carried out on wild-type and site directed mutated hKv1.5 channels stably expressed on Ltk(-) cells using the whole-cell patch-clamp. RESULTS: At 35 mM [K+](i) the voltage-dependent unblock produced by 500 microM benzocaine was preserved at both 4 and 140 mM [K+](o). Mutations located in the inner mouth of the pore (T477S, T505A, L508M and V512M) abolished the agonist but increased the blocking effects of benzocaine. Intracellular application of tetraethylammonium (3 mM) abolished the 'agonist' effects whereas the blocking effects of benzocaine remained unaltered. Block induced by benzocaine and intracellular tetraethylammonium was additive. In contrast, the combination of benzocaine and bupivacaine (>25 microM) produced less blockade than bupivacaine alone. However, mutation of the extracellular residue R485Y did not modify the effects of benzocaine. Extracellular application of tetraethylammonium (100 mM) did not modify the agonist effects of benzocaine, but abolished the voltage- and time-dependence of benzocaine-induced block. CONCLUSIONS: The results suggested that 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. Furthermore, benzocaine and extracellular K(+) interact to modify the voltage-dependence of channel opening.
OBJECTIVES: It has been demonstrated that at nanomolar concentrations benzocaine increased, whereas at micromolar concentrations, it blocked hKv1.5 channels in a voltage-dependent manner and modified the voltage-dependence of channel activation. The present study was undertaken to localize the putative binding sites involved in the 'agonists' and blocking effects of benzocaine. METHODS: Experiments were carried out on wild-type and site directed mutated hKv1.5 channels stably expressed on Ltk(-) cells using the whole-cell patch-clamp. RESULTS: At 35 mM [K+](i) the voltage-dependent unblock produced by 500 microM benzocaine was preserved at both 4 and 140 mM [K+](o). Mutations located in the inner mouth of the pore (T477S, T505A, L508M and V512M) abolished the agonist but increased the blocking effects of benzocaine. Intracellular application of tetraethylammonium (3 mM) abolished the 'agonist' effects whereas the blocking effects of benzocaine remained unaltered. Block induced by benzocaine and intracellular tetraethylammonium was additive. In contrast, the combination of benzocaine and bupivacaine (>25 microM) produced less blockade than bupivacaine alone. However, mutation of the extracellular residue R485Y did not modify the effects of benzocaine. Extracellular application of tetraethylammonium (100 mM) did not modify the agonist effects of benzocaine, but abolished the voltage- and time-dependence of benzocaine-induced block. CONCLUSIONS: The results suggested that 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. Furthermore, benzocaine and extracellular K(+) interact to modify the voltage-dependence of channel opening.