Fast- or Slow-inactivated State Preference of Na+ Channel Inhibitors: A Simulation and Experimental Study

Sodium channels are one of the most intensively studied drug targets. Sodium channel inhibitors (e.g., local anesthetics, anticonvulsants, antiarrhythmics and analgesics) exert their effect by stabilizing an inactivated conformation of the channels. Besides the fast-inactivated conformation, sodium channels have several distinct slow-inactivated conformational states. Stabilization of a slow-inactivated state has been proposed to be advantageous for certain therapeutic applications. Special voltage protocols are used to evoke slow inactivation of sodium channels. It is assumed that efficacy of a drug in these protocols indicates slow-inactivated state preference. We tested this assumption in simulations using four prototypical drug inhibitory mechanisms (fast or slow-inactivated state preference, with either fast or slow binding kinetics) and a kinetic model for sodium channels. Unexpectedly, we found that efficacy in these protocols (e.g., a shift of the “steady-state slow inactivation curve”), was not a reliable indicator of slow-inactivated state preference. Slowly associating fast-inactivated state-preferring drugs were indistinguishable from slow-inactivated state-preferring drugs. On the other hand, fast- and slow-inactivated state-preferring drugs tended to preferentially affect onset and recovery, respectively. The robustness of these observations was verified: i) by performing a Monte Carlo study on the effects of randomly modifying model parameters, ii) by testing the same drugs in a fundamentally different model and iii) by an analysis of the effect of systematically changing drug-specific parameters. In patch clamp electrophysiology experiments we tested five sodium channel inhibitor drugs on native sodium channels of cultured hippocampal neurons. For lidocaine, phenytoin and carbamazepine our data indicate a preference for the fast-inactivated state, while the results for fluoxetine and desipramine are inconclusive. We suggest that conclusions based on voltage protocols that are used to detect slow-inactivated state preference are unreliable and should be re-evaluated.Author Summary: Sodium channels are the key proteins for action potential firing in most excitable cells. Inhibitor drugs prevent excitation (local anesthetics), regulate excitability (antiarrhythmics), or prevent overexcitation (antiepileptic, antispastic and neuroprotective drugs) by binding to the channel and keeping it in one of the inactivated channel conformations. Sodium channels have one fast- and several slow-inactivated conformations (states). The specific stabilization of slow-inactivated states have been proposed to be advantageous in certain therapeutic applications. The question of whether individual drugs stabilize the fast or the slow-inactivated state is studied using specific voltage protocols. We tested the reliability of conclusions based on these protocols in simulation experiments using a model of sodium channels, and we found that fast- and slow-inactivated state-stabilizing drugs could not be differentiated. We suggested a method by which the state preference of at least a subset of individual drugs could be determined and tried the method in electrophysiology experiments with five individual drugs. Three of the drugs (lidocaine, phenytoin and carbamazepine) were classified as fast-inactivated state-stabilizers, while the state preference of fluoxetine and desipramine was found to be undeterminable by this method.