Inhalational anaesthetics and n-alcohols share a site of action in the neuronal Shaw2 Kv channel.
OBJECTIVE
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(+) (K(v)) channel to ask whether the inhalational anaesthetic halothane and n-alcohols share a binding site near the activation gate of the channel.


METHODS
Focusing on activation gate mutations that affect channel modulation by n-alcohols, we investigated n-alcohol-sensitive and n-alcohol-resistant K(v) 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.


RESULTS
Shaw2 K(v) channels are reversibly inhibited by halothane in a dose-dependent and saturable manner (K(0.5)= 400 microM; n(H)= 1.2). Also, discrete mutations in the channel's S4S5 linker are sufficient to reduce or confer inhibition by halothane (Shaw2-T330L and K(v)3.4-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
These observations strongly support a molecular model of a general anaesthetic binding site in the Shaw2 K(v) channel. This site may involve the amphiphilic interface between the S4S5 linker and the S6 segment, which plays a pivotal role in K(v) channel activation.
Department of Pathology, Anatomy and Cell Biology, Jefferson Medical College of Thomas Jefferson University, Philadelphia, PA, USA.
Aditya
A
Bhattacharji
Nathan
N
Klett
Ramon Christopher V
RC
Go
Manuel
M
Covarrubias
D000438:Alcohols / Q000494:pharmacology*; D018685:Anesthetics, Inhalation / Q000494:pharmacology*; D000818:Animals; D006221:Halothane / Q000494:pharmacology*; D009154:Mutation; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D051670:Shaw Potassium Channels / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis
D000438:Alcohols; D018685:Anesthetics, Inhalation; D051670:Shaw Potassium Channels; D006221:Halothane