The Sensitivity of G Protein-activated K+ Channels toward Halothane Is Essentially Determined by the C Terminus

Sergej Milovic,Bibiane Steinecker-Frohnwieser,Wolfgang Schreibmayer,Lukas G Weigl

doi:10.1074/jbc.m403448200

Abstract

G protein-activated K(+) channels (GIRKs or Kir3.x) are targets for the volatile anesthetic, halothane. When coexpressed with the m(2) acetylcholine (ACh) receptor in Xenopus oocytes, agonist-activated GIRK1(F137S)- and GIRK2-mediated currents are inhibited by halothane, whereas in the absence of ACh, high concentrations of halothane induce GIRK1(F137S)-mediated currents. To elucidate the molecular mechanism of halothane action on GIRK currents of different subunit compositions, we constructed deletion mutants of GIRK1(F137S) (GIRK1(Delta363*)) and GIRK2 (GIRK2(Delta356)) lacking the C-terminal ends, as well as chimeric GIRK channels. Mutated GIRK channels showed normal currents when activated by ACh but exhibited different pharmacological properties toward halothane. GIRK2(Delta356) showed no sensitivity against the inhibitory action of halothane but was activated by halothane in the absence of an agonist. GIRK1(Delta363*) was activated by halothane more efficiently. Currents mediated by chimeric channels were inhibited by anesthetic concentrations that were at least 30-fold lower than those necessary to decrease GIRK2 wild type currents. Glutathione S-transferase pulldown experiments did not show displacement of bound Gbetagamma by halothane, indicating that halothane does not interfere with Gbetagamma binding. Single channel experiments revealed an influence of halothane on the gating of the channels: The agonist-induced currents of GIRK1 and GIRK2, carried mainly by brief openings, were inhibited, whereas higher concentrations of the anesthetic promoted long openings of GIRK1 channels. Because the C terminus is crucial for these effects, an interaction of halothane with the channel seems to be involved in the mechanism of current modulation.

Highlights

G protein-activated K؉ channels (GIRKs or Kir3.x) are targets for the volatile anesthetic, halothane
Besides ion channels, which have been shown to be modulated by anesthetics (2), other elements of signal transduction such as receptors and G proteins may be involved in general anesthesia
The activation of G protein-activated inwardly rectifying Kϩ (GIRK) channels leads to a decrease in heart rate after the release of acetylcholine from the vagus nerve, and in the central nervous system GIRK channels play an important role in the mediation of opioidand ethanol-induced analgesia (9 –12)

Summary

Introduction

G protein-activated K؉ channels (GIRKs or Kir3.x) are targets for the volatile anesthetic, halothane. GIRK2 channels are not activated by halothane at the basal level but are most sensitive to the inhibitory action of halothane when activated by an agonist These effects are independent from the receptor coexpressed and specific for the GIRK channels (14). From our previous experiments (14) we concluded that halothane was able to activate the GIRK1 channel, probably by increasing its affinity to G␤␥, and that the inhibition of agonist-activated GIRK channels was because of the impairment of the G protein signaling cascade. This is consistent with the findings of various other laboratories that demonstrate the inhibition of signaling through G␣i by halothane (15–18). This structure is exposed to the cytoplasm and is believed to encom-

Methods

Results

Conclusion