title: Block of inactivation-deficient Na+ channels by local anesthetics in stably transfected mammalian cells: evidence for drug binding along the activation pathway.
abstract: 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 microM (n = 5) and 81.7 +/- 10.6 microM (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 microM benzocaine, rapid time-dependent block (tau approximately 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.
authors.affiliation: Department of Biology, State University of New York at Albany, NY 12222, USA.
authors.forename: Sho-Ya
authors.initials: SY
authors.lastname: Wang
authors.forename: Jane
authors.initials: J
authors.lastname: Mitchell
authors.forename: Edward
authors.initials: E
authors.lastname: Moczydlowski
authors.forename: Ging Kuo
authors.initials: GK
authors.lastname: Wang
mesh_terms: D000779:Anesthetics, Local / Q000494:pharmacology*; D000818:Animals; D001566:Benzocaine / Q000494:pharmacology*; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D016503:Drug Delivery Systems / Q000379:methods; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects* / Q000502:physiology*; D007668:Kidney / Q000187:drug effects / Q000502:physiology*; D008012:Lidocaine / Q000494:pharmacology*; D008564:Membrane Potentials / Q000187:drug effects; D009124:Muscle Proteins / Q000187:drug effects* / Q000502:physiology*; D051381:Rats; D015222:Sodium Channels / Q000187:drug effects* / Q000502:physiology*; D013329:Structure-Activity Relationship; D014162:Transfection / Q000379:methods
chemical_list: D000779:Anesthetics, Local; D009124:Muscle Proteins; C466180:Scn4a protein, rat; D015222:Sodium Channels; D008012:Lidocaine; D001566:Benzocaine