Block of Inactivation-deficient Na⁺ Channels by Local Anesthetics in Stably Transfected Mammalian Cells : Evidence for Drug Binding Along the Activation Pathway

According to the classic modulated receptor hypothesis, local anesthetics (LAs) such as benzocaine and lidocaine bind preferentially to fast-inactivated Na⁺ channels with higher affinities. However, an alternative view suggests that activation of Na⁺ channels plays a crucial role in promoting high-affinity LA binding and that fast inactivation per se is not a prerequisite for LA preferential binding. We investigated the role of activation in LA action in inactivation-deficient rat muscle Na⁺ channels (rNav1.4-L435W/L437C/A438W) expressed in stably transfected Hek293 cells. The 50% inhibitory concentrations (IC₅₀) for the open-channel block at +30 mV by lidocaine and benzocaine were 20.9 ± 3.3 μM (n = 5) and 81.7 ± 10.6 μM (n = 5), respectively; both were comparable to inactivated-channel affinities. In comparison, IC₅₀ values for resting-channel block at −140 mV were >12-fold higher than those for open-channel block. With 300 μM benzocaine, rapid time-dependent block (τ ≈ 0.8 ms) of inactivation-deficient Na⁺ currents occurred at +30 mV, but such a rapid time-dependent block was not evident at −30 mV. The peak current at −30 mV, however, was reduced more severely than that at +30 mV. This phenomenon suggested that the LA block of intermediate closed states took place notably when channel activation was slow. Such closed-channel block also readily accounted for the LA-induced hyperpolarizing shift in the conventional steady-state inactivation measurement. Our data together illustrate that the Na⁺ channel activation pathway, including most, if not all, transient intermediate closed states and the final open state, promotes high-affinity LA binding.