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 (IC 50 ) for the open-channel block at 30 mV by lidocaine and benzocaine were 20.9 3.3 M ( n 5) and 81.7 10.6 M ( n 5), respectively; both were comparable to inactivated-channel affinities. In comparison, IC 50 values for resting-channel block at 140 mV were 12-fold higher than those for open-channel block. With 300 M benzocaine, rapid time-dependent block ( 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. key words: sodium channel • fast inactivation • persistent sodium currents • local anesthetics • modulated receptor hypothesis I N T R O D U C T I O N The in vivo targets of local anesthetics (LAs) are voltagegated Na channels responsible for the generation of action potentials in excitable membranes (Hille, 2001). Molecular cloning has thus far identified nine Na channel -subunit isoforms in mammals. The -subunit isoforms contain four repeated domains (D1–D4), each with six transmembrane domains (S1–S6) and can be functionally expressed in frog oocytes or in mammalian expression systems (Catterall, 2000). LAs interact with residues in the middle of D4S6, D3S6, and D1S6 segments, probably situated within the inner cavity of the Na permeation pathway (Ragsdale et al., 1994; Wang et al., 1998, 2000; Yarov-Yarovoy et al., 2001, 2002). The block of Na currents by lidocaine and benzocaine has been studied extensively. Lidocaine contains a tertiary amine group, which can be protonated in the aqueous solution. In contrast, benzocaine is a neutral LA. Lidocaine not only elicits a tonic block of Na currents when infrequently stimulated but also produces an additional use-dependent block during repetitive pulses (Hille, 2001). On the other hand, benzocaine fails to produce any use-dependent block. As a neutral drug, benzocaine may escape too rapidly during the interpulse to accumulate such a block. However, both drugs significantly shift the steady-state inactivation of Na channels to the hyperpolarizing direction as if LAs stabilize the inactivated state. Hille (1977) proposed a modulated receptor hypothesis to account for the complicated block of LAs. His hypothesis states that the LA receptor changes its conformation during state transitions and that the fast-inactivated state has a higher LA affinity than does the resting state. A similar hypothesis (Hondeghem and Katzung 1977, 1984) suggested that a class 1 antiarrhythmic agent, lidocaine, may additionally block open cardiac Na channels with a higher affinity. To date, the proof of the high-affinity open-channel block by LAs remains controversial. Several pieces Address correspondence to Ging Kuo Wang, Dept. of Anesthesia, Brigham and Women’s Hospital, 75 Francis St., Boston, MA 02115. Fax: 617730-2801; email:wang@zeus.bwh.harvard.edu Abbreviation used in this paper: LA, local anesthetic. on Jne 3, 2017 D ow nladed fom Published November 15, 2004