Inhalational anaesthetics and n‐alcohols share a site of action in the neuronal Shaw2 K_v channel

Abstract

Background and purpose: Neuronal ion channels are key targets of general anaesthetics and alcohol, and binding of these drugs to pre-existing and relatively specific sites is thought to alter channel gating. However, the underlying molecular mechanisms of this action are still poorly understood. Here, we investigated the neuronal Shaw2 voltage-gated K + (Kv) channel to ask whether the inhalational anaesthetic halothane and n-alcohols share a binding site near the activation gate of the channel. Experimental approach: Focusing on activation gate mutations that affect channel modulation by n-alcohols, we investigated n-alcohol-sensitive and n-alcohol-resistant Kv channels heterologously expressed in Xenopus oocytes to probe the functional modulation by externally applied halothane using two-electrode voltage clamping and a gas-tight perfusion system. Key results: Shaw2 Kv channels are reversibly inhibited by halothane in a dose-dependent and saturable manner (K0.5 = 400 mM; nH = 1.2). Also, discrete mutations in the channel's S4S5 linker are sufficient to reduce or confer inhibition by halothane (Shaw2-T330L and Kv3.4-G371I/T378A respectively). Furthermore, a point mutation in the S6 segment of Shaw2 (P410A) converted the halothane-induced inhibition into halothane-induced potentiation. Lastly, the inhibition resulting from the co-application of n-butanol and halothane is consistent with the presence of overlapping binding sites for these drugs and weak binding cooperativity. Conclusions and implications:These observations strongly support a molecular model of a general anaesthetic binding site in the Shaw2 Kv channel. This site may involve the amphiphilic interface between the S4S5 linker and the S6 segment, which plays a pivotal role in Kv channel activation.

Insight into the modulation of Shaw2 Kv channels by general anesthetics: Structural and functional studies of S4–S5 linker and S6 C-terminal peptides in micelles by NMR

“…Upon depolarization of the membrane potential, the voltage sensors adopt their “up” conformation and pull on L45, which acts as the “handle” that actively opens the activation gate through its interaction with S6c. 1-Butanol and other inhaled anesthetics may dislodge this interaction and, consequently, the channel remains closed [25]. …”

“…The Shaw2 channel is selectively inhibited by 1-alkanols and halothane at pharmacologically relevant concentrations [23–25]. The action of the inhibitors is consistent with binding to an intracellular site and the stabilization of the channel’s close state [26].…”

Novel Activation of Voltage-gated K+ Channels by Sevoflurane

“…Electrophysiology-Whole-oocyte currents were recorded at room temperature (20 -23°C) under two-electrode voltageclamp conditions (OC-725C, Warner Instrument, Hamden, CT) according to established procedures (12,31,33). Briefly, oocytes were bathed in ND96 and electrophysiological recordings were performed using borosilicate microelectrodes with tip resistances of 0.2-0.5 M⍀ when filled with 3 M KCl.…”

“…Reagents-Volatile anesthetic stocks were diluted in in ND96 as previously described (12,33). Final dilutions of all drugs were prepared immediately prior to use.…”

“…Currents by Sevoflurane-Previous work with K-Shaw2 and halogenated anesthetics (12,33,37) prompted us to examine other clinically relevant general anesthetics. Thus, we constructed concentration-response relations to compare the inhibition of K-Shaw2 by 1-butanol and halothane to the effects of chloroform, isoflurane, desflurane, sevoflurane, and propofol FIGURE 1.…”