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

Abstract

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.

Mechanism of Modification, by Lidocaine, of Fast and Slow Recovery from Inactivation of Voltage-Gated Na+ Channels

“…Values during lidocaine exposure are compared to the same protocol under control conditions. because of the difficulty in adjusting these protocols to the complex mutation-induced alterations in the time course of recovery (Karoly et al, 2010). Mutation-Induced Changes in Recovery from Inactivation.…”

Inhibition of Neuronal Voltage-Gated Sodium Channels by Brilliant Blue G

“…In addition, as noted by Karoly et al (2010), models in which BBG binding was exclusive to the fast-inactivated state could predict the concentration-dependent shift in the apparent voltage-dependence of slow inactivation (Fig. 6A), because drug-bound fast-inactivated channels recover slowly and can mimic enhanced slow inactivation.…”

“…Most significantly, the model did not produce as large a difference as was present experimentally in the kinetics of development of block by 3 M BBG with a conditioning prepulse to 0 mV compared with Ϫ50 mV. The model of a single fast-inactivated state and a single slow-inactivated state is highly simplified compared with the actual behavior of sodium channels, which have multiple fast-inactivated states reached from different closed states (Kuo and Bean, 1994a) and very likely multiple slow-inactivated states as well (Karoly et al, 2010). In addition, for simplicity, we connected the slow-inactivated state sequentially to the fastinactivated state, so that channels are in one or the other but not both.…”

“…As noted by Karoly et al (2010), it is difficult to distinguish between models in which drugs bind with high affinity to slow-inactivated states and models in which drugs bind only to fast-inactivated states, but with slow kinetics. Our model includes binding to both fast-inactivated channels and slow-inactivated channels, and both seemed to be required for adequate modeling of the experimental results.…”

“…In addition, for simplicity, we connected the slow-inactivated state sequentially to the fastinactivated state, so that channels are in one or the other but not both. In fact, however, the fast-inactivation gate can move independently of whether channels are slow-inactivated (Vedantham and Cannon, 1998), which would give considerably more complex models (Vedantham and Cannon, 1998;Karoly et al, 2010). Models with additional fast-or slow-inactivated states might better predict the much faster kinetics of BBG action during conditioning pulses to 0 mV compared with Ϫ50 mV.…”