Modulation of Inactivation Kinetics of the Bacterial Sodium Channel Nachbac Suggests a Complex Mode of Inhibition by Isoflurane

Abstract

The bacterial sodium channel NaChBac is a prokaryotic ancestor of eukaryotic voltage-gated sodium channels (Nav), which are critical for action potential generation and propagation in nervous and cardiac tissue. Like all mammalian Nav isoforms examined thus far, NaChBac is inhibited by clinically relevant concentrations of the inhaled volatile anesthetic isoflurane, and inhibition is accompanied by enhancement of slow or C-type inactivation (Ouyang et al., 2007). However, a detailed mechanistic explanation of the interplay between C-type inactivation and isoflurane inhibition is lacking. To shed light on the relationship between inactivation and inhibition, we introduced point mutations known to alter inactivation in NaChBac (G219A, G219P, G229A, and S195E), expressed the channels in HEK293FT cells, and applied isoflurane during whole-cell patch clamp recording. Preliminary data support the idea that isoflurane acts by multiple mechanisms invovling multiple sites on NaChBac. Channel mutations that enhance inactivation show greater current reduction by isoflurane, suggesting that isoflurane binds more favorably to inactivated channels. However, isoflurane exhibits both tonic and use-dependent block of the essentially non-inactivating NaChBac G219P mutant, indicating that binding also occurs in the closed/resting and open/conducting states. A detailed biophysical and pharmacological profile of these NaChBac inactivation variants together with a recent molecular dynamics simulation study showing isoflurane interacting with NaChBac at three distinct binding sites (Carnavale et al, 2013) supports multiple mechanisms of state-dependent inhibtion. Such functional data help clarify the complex pharmacological effects of volatile anesthetics on Nav channels, and contribute to better understanding of C-type inactivation in these proteins. Supported by NIH grant GM 58055 (RS and HH) and German Research Foundation fellowship 5767/1-1 (TM).