G Protein βγ Gating Confers Volatile Anesthetic Inhibition to Kir3 Channels

Abstract

G protein-activated inwardly rectifying potassium (GIRK or Kir3) channels are directly gated by the ␤␥ subunits of G proteins and contribute to inhibitory neurotransmitter signaling pathways. Paradoxically, volatile anesthetics such as halothane inhibit these channels. We find that neuronal Kir3 currents are highly sensitive to inhibition by halothane. Given that Kir3 currents result from increased G␤␥ available to the channels, we asked whether reducing available G␤␥ to the channel would adversely affect halothane inhibition. Remarkably, scavenging G␤␥ using the C-terminal domain of ␤-adrenergic receptor kinase (c␤ARK) resulted in channel activation by halothane. Consistent with this effect, channel mutants that impair G␤␥ activation were also activated by halothane. A single residue, phenylalanine 192, occupies the putative G␤␥ gate of neuronal Kir3.2 channels. Mutation of Phe-192 at the gate to other residues rendered the channel non-responsive, either activated or inhibited by halothane. These data indicated that halothane predominantly interferes with G␤␥-mediated Kir3 currents, such as those functioning during inhibitory synaptic activity. Our report identifies the molecular correlate for anesthetic inhibition of Kir3 channels and highlights the significance of these effects in modulating neurotransmitter-mediated inhibitory signaling.Ion channels that control the excitability of neuronal conduction pathways are important targets for halogenated volatile anesthetics (HVAs) 4 (1, 2). HVAs such as halothane enhance the activity of inhibitory GABA A and glycine receptors and inhibit excitatory channels such as glutamate and nicotinic receptors (3). Several two-pore domain K (K 2 P) channels are activated by HVAs (4 -6). G protein-activated inwardly rectifying potassium (GIRK or Kir3) channels are also involved in inhibitory neurotransmission (7). Although ethanol and chloroform activate Kir3 channels (8, 9), paradoxically HVAs inhibit Kir3 channels (10, 11). Therefore, Kir3 channels present a rare case where an HVA such as halothane impairs inhibitory signaling by an ion channel.The exact mechanism for modulation of ion channels by volatile anesthetics is not fully clear (2). Two residues in the TM2 and TM3 of GABA A participate in regulating the effects of enflurane (12). In nicotinic receptors, sites of action for halothane on tyrosine residues near the channel pore were identified (13). The transmembrane domains in AMPA and NMDA receptors play critical roles in their anesthetics and ethanol sensitivity (14,15). Several sites on various K 2 P channels have been identified that are involved in anesthetic sensitivity (5, 16).Inhibitory neurotransmitters, exemplified by GABA, stimulate G␣ i/o -coupled receptors (17, 18) and liberate G␤␥ that directly activates Kir3 channels (19). Kir3 channels mediate slow inhibitory post-synaptic currents in central neurons, limiting neuronal excitability (7). Kir3-null mice show increased seizure susceptibility (20), hyperalgesia (21), and reduced neurotransmitter-, morphine...

Structural Insights into GIRK Channel Function

“…2) and demonstrated that mutation of this residue to the equivalent residue in halothane-insensitive Kir2.1, a methionine, resulted in reversal of halothane inhibition. Similar results were also demonstrated for a mutation that inhibited Gβγ activation (GIRK2 L273I) (Styer et al, 2010). Several other anesthetics acted as agonist or antagonists toward GIRK channels.…”

“…GIRK1/GIRK4 channel activation by halothane appears to involve a Gβγ-dependent enhancement of basal current, while it also inhibits agonist-induced current (Weigl & Schreibmayer, 2001). The TM domains and carboxy-terminus were shown to mediate halothane interactions (Milovic, SteineckerFrohnwieser, Schreibmayer, & Weigl, 2004;Styer et al, 2010;Yamakura et al, 2001). Chimeric studies probing the determinants of halothane sensitivity implicated the phenylalanine at the helix bundle crossing (GIRK2 F192; Fig.…”

When anaesthetics collide: antagonism of one agent by another?

“…However, these receptors may not be anaesthetically relevant, as they constitute < 10% of GABA subtypes, are found almost exclusively in cerebellar granule cells and classed as ‘benzodiazepine‐insensitive’. Similar partial agonist behaviour for halothane has been reported for a subunit of an inwardly rectifying K+ channel and Jonsson Fagerlund et al describe propofol as a partial agonist at the α2β2γ2 GABA‐A receptor . Nitrous oxide can be regarded as a partial agonist, as its MAC is > 100%, so it never reaches its full efficacy for consciousness or for analgesic properties (the latter action exerted via opiate receptors) .…”

Synergistic Roles for G-protein γ3 and γ7 Subtypes in Seizure Susceptibility as Revealed in Double Knock-out Mice

“…After plating onto 12-mm polylysine-treated coverslips, patch clamp analyses were performed on neurons in culture between 11 and 14 days without prior knowledge of the genotype. Currents from G-protein-sensitive inwardly rectifying potassium (GIRK, Kir3) channels were measured as described previously (14). Briefly, neurons were constantly voltage-clamped at Ϫ60 mV, recorded using a Multiclamp700B amplifier, digitized with a Digidata 1322B, sampled at 4 kHz, low pass-filtered at 1 kHz, and collected using pClamp9.2 (all from Molecular Devices).…”