Reduced inhibition of cortical glutamate and GABA release by halothane in mice lacking the K+ channel, TREK-1

Abstract

Background and purpose: Deletion of TREK-1, a two-pore domain K(+) channel (K(2P)) activated by volatile anaesthetics, reduces volatile anaesthetic potency in mice, consistent with a role for TREK-1 as an anaesthetic target. We used TREK-1 knockout mice to examine the presynaptic function of TREK-1 in transmitter release and its role in the selective inhibition of glutamate vs GABA release by volatile anaesthetics.

Experimental approach: The effects of halothane on 4-aminopyridine-evoked and basal [(3)H]glutamate and [(14)C]GABA release from cerebrocortical nerve terminals isolated from TREK-1 knockout (KO) and littermate wild-type (WT) mice were compared. TREK-1 was quantified by immunoblotting of nerve terminal preparations.

Key results: Deletion of TREK-1 significantly reduced the potency of halothane inhibition of 4-aminopyridine-evoked release of both glutamate and GABA without affecting control evoked release or the selective inhibition of glutamate vs GABA release. TREK-1 deletion also reduced halothane inhibition of basal glutamate release, but did not affect basal GABA release.

Conclusions and implications: The reduced sensitivity of glutamate and GABA release to inhibition by halothane in TREK-1 KO nerve terminals correlates with the reduced anaesthetic potency of halothane in TREK-1 KO mice observed in vivo. A presynaptic role for TREK-1 was supported by the enrichment of TREK-1 in isolated nerve terminals determined by immunoblotting. This study represents the first evidence for a link between an anaesthetic-sensitive 2-pore domain K(+) channel and presynaptic function, and provides further support for presynaptic mechanisms in determining volatile anaesthetic action.

Figure 1

Halothane inhibits 4AP-evoked glutamate (a) and GABA (b) release from wild-type littermate (WT) and TREK-1 knockout (KO) mouse cortical nerve terminals. Data are presented binned with curve fits and statistical analyses performed on individually measured halothane concentrations. Sum ΔFR in the presence (+Ca²⁺) or absence (−Ca²⁺) of Ca²⁺, and halothane concentrations are presented as mean±s.e.m. Control data are presented as mean±s.d. Shaded areas highlight the clinical concentration range between 0.5 and 2 times MAC for WT mouse (0.24 mM; Heurteaux et al., 2004). Halothane incompletely inhibited 4AP-evoked glutamate release (Imax≠0) from WT nerve terminals in the presence or absence of extracellular Ca²⁺ (P<0.0001 and P<0.0001, respectively). Halothane completely (Imax=0) inhibited 4AP-evoked GABA release from WT nerve terminals in the presence (P=0.39) or absence (P=0.40) of Ca²⁺. In TREK-1 KO nerve terminals, in the presence or absence of Ca²⁺, halothane also incompletely inhibited 4AP-evoked glutamate release (P=0.005 and P=0.005, respectively), and completely inhibited 4AP-evoked GABA release (P=0.79 and P=0.83, respectively). IC₅₀ values are shown in Figure 2.

Figure 2

Halothane inhibition of 4AP-evoked glutamate and GABA release from wild-type littermate (WT) and TREK-1 knockout (KO) mouse cortical nerve terminals. IC₅₀ values (mean±s.e.m.) were compared between glutamate and GABA release (^***P<0.001), and between WT and KO mice (^†††P<0.001) using the F-test (Prism 4.0). Shaded area highlights the clinical concentration range (0.5–2 times MAC) for WT mice (0.24 mM; Heurteaux et al., 2004).

Figure 3

Halothane inhibits residual tetrodotoxin-insensitive 4AP-evoked glutamate release. At a nearly maximal inhibitory concentration (∼2 times MAC for WT mouse), halothane (Hal) inhibited 4AP-evoked glutamate and GABA release in the presence of a saturating concentration of tetrodotoxin (TTX, 1 μM) in WT (0.71±0.04 mM) and KO (0.75±0.06 mM) mice, compared to halothane (0.76±0.03 and 0.75±0.04 mM, respectively) or tetrodotoxin alone, in the presence of Ca²⁺. Statistical analysis of mean±s.e.m. values was performed by one-way ANOVA, with Bonferroni post hoc testing between respective control (^***P<0.001) and between indicated groups (^†P<0.05, ^†††P<0.001).

Figure 4

Inhibition of basal glutamate release by halothane is reduced in TREK-1 knockout mice. Basal release of glutamate (a) and GABA (b) in the absence of Ca²⁺ from wild-type littermate (WT) or TREK-1 knockout (KO) mouse cortical nerve terminals was determined in the absence (control, n=5) or presence of halothane (0.99±0.06 mM, n=6, and 1.03±0.03 mM, n=6, respectively). Halothane effects on basal release (Emax) were compared by curve fitting (Prism 4.0). Prestimulus baseline release was standardized and is denoted by a dotted line. ^***P<0.001 vs WT.

Figure 5

TREK-1 immunoreactivity is enriched in mouse cortical nerve terminals. Mouse cortex was homogenized (crude homogenate) and further purified through sucrose gradients (synaptosomes). Representative immunoblot shows immunoreactive TREK-1 band at 67 kDa (40 μg protein per lane). Quantification by scanning of the immunoblots indicated a 1.8-fold enrichment of TREK-1 in cortical synaptosomes (n=4) vs crude homogenate (n=4; P<0.01).