The actions of ether, alcohol and alkane general anaesthetics on GABAA and glycine receptors and the effects of TM2 and TM3 mutations

Abstract

The actions of 13 general anaesthetics (diethyl ether, enflurane, isoflurane, methoxyflurane, sevoflurane, chloral hydrate, trifluoroethanol, tribromoethanol, tert-butanol, chloretone, brometone, trichloroethylene, and alpha-chloralose) were studied on agonist-activated Cl(-) currents at human GABA(A) alpha(2)beta(1), glycine alpha(1), and GABA(C) rho(1) receptors expressed in human embryonic kidney 293 cells. All 13 anaesthetics enhanced responses to submaximal (EC(20)) concentrations of agonist at GABA(A) and glycine receptors, except alpha-chloralose, which did not enhance responses at the glycine alpha(1) receptor. None of the anaesthetics studied potentiated GABA responses at the GABA(C) rho(1) receptor. Potentiation of submaximal agonist currents by the anaesthetics was studied at GABA(A) and glycine receptors harbouring mutations in putative transmembrane domains 2 and 3 within GABA(A) alpha(2), beta(1), or glycine alpha(1) receptor subunits: GABA(A) alpha(2)(S270I)beta(1), alpha(2)(A291W)beta(1), alpha(2)beta(1)(S265I), and alpha(2)beta(1)(M286W); glycine alpha(1)(S267I) and alpha(1)(A288W). For all anaesthetics studied except alpha-chloralose, at least one of the mutations above abolished drug potentiation of agonist responses at GABA(A) and glycine receptors. alpha-Chloralose produced efficacious direct activation of the GABA(A) alpha(2)beta(1) receptor (a 'GABA-mimetic' effect). The other 12 anaesthetics produced minimal or no direct activation of GABA(A) and glycine receptors. A non-anaesthetic isomer of alpha-chloralose, beta-chloralose, was inactive at GABA(A) and glycine receptors and did not antagonize the actions of alpha-chloralose at GABA(A) receptors. The implications of these findings for the molecular mechanisms of action of general anaesthetics at GABA(A) and glycine receptors are discussed.

Figure 1

Chemical structures of the anaesthetic compounds investigated in this study.

Figure 2

Potentiation of current responses to EC₂₀ concentrations of GABA at wild-type human GABA_A α₂β₁ receptors by sevoflurane (A), methoxyflurane (B), and chloral hydrate (C). Note the ‘rebound' current (arrow) following washout of 5 mM sevoflurane. Both sevoflurane and methoxyflurane directly activate the GABA_A α₂β₁ receptor, particulary noticeable during the pre-application of 5 mM sevoflurane or methoxyflurane. Traces shown are individual recordings from HEK 293 cells transfected with cDNAs encoding the GABA_A α₂ and β₁ receptor subunits.

Figure 3

Potentiation of current responses to EC₂₀ concentrations of glycine (Gly) at wild-type human glycine α₁ receptors by sevoflurane (A), methoxyflurane (B), and chloral hydrate (C). Sevoflurane (5 mM) produces slight direct activation of the glycine α₁ receptor. Traces shown are individual recordings from HEK 293 cells transfected with cDNA encoding the glycine α₁ receptor subunit.

Figure 4

α-Chloralose produces potentiation of GABA responses and direct activation at the GABA_A α₂β₁ receptor, yet inhibits submaximal glycine responses at the glycine α₁ receptor. (A) α-Chloralose potentiates current responses to EC₂₀ concentrations of GABA at the GABA_A α₂β₁ receptor. Direct activation is evident during the pre-application of 50 and 200 μM α-chloralose. (B) α-Chloralose directly activates the GABA_A α₂β₁ receptor. The first four traces are for α-chloralose applied in the absence of any GABA in the perfusion system. The response to a maximal concentration of GABA (500 μM) is shown for comparison. Note the slow time course of the α-chloralose-mediated currents. (C) α-Chloralose, at concentrations greater than 100 μM, inhibits current responses to EC₂₀ concentrations of glycine (Gly) at the glycine α₁ receptor. Traces shown are individual recordings from HEK 293 cells transfected with cDNAs encoding the GABA_A α₂ and β₁ receptor subunits (A,B) or the glycine α₁ receptor subunit (C).

Figure 5

Pooled concentration-response relationships for (A) sevoflurane, (B) diethyl ether, (C) chloral hydrate, and (D) α-chloralose modulation of currents in response to submaximal (EC₂₀) concentrations of agonist at wild-type GABA_A α₂β₁ and glycine α₁ receptors. Note the lack of effect of α-chloralose at the glycine α₁ receptor at concentrations ⩽0.1 mM with inhibition of glycine responses evident at 0.2, 0.5, and 1 mM. See Table 1 for EC₅₀, Hill slope, E_max, and n_cells values.

Figure 6

Mutations in TM2 or TM3 of the GABA_A α₂ subunit abolish potentiation of responses to EC₂₀ concentrations of GABA by the ether anaesthetics sevoflurane (SEV), methoxyflurane (MXY), and diethyl ether (DEE). (A,B) Sevoflurane (1 mM) and methoxyflurane (2 mM) fail to potentiate submaximal GABA currents, while diethyl ether (50 mM) inhibits GABA responses at GABA_A α₂(S270I)β₁ and GABA_A α₂(A291W)β₁ receptors. (C) In contrast, sevoflurane, methoxyflurane, and diethyl ether potentiate submaximal GABA responses at the GABA_A α₂β₁(S265I) receptor. Note that the concentrations of GABA required to produce 20% of a maximal response (i.e., the EC₂₀ concentration) are different for each of the receptors depicted in (A), (B), and (C). Some of the receptors containing mutated subunits have apparent affinities for GABA that differ from that at the wild-type GABA_A α₂β₁ receptor. Traces shown are individual recordings from HEK 293 cells transfected with cDNAs encoding the indicated receptor subunit combinations.

Figure 7

Summary of the effects of mutations in TM2 or TM3 of GABA_A α₂ or β₁ receptor subunits on potentiation of GABA responses by general anaesthetics. The general anaesthetics are sorted into (A) ethers, (B) primary alcohols along with trichloroethylene and propofol, and (C) tertiary alcohols and α-chloralose. The ordinate depicts percentage change of a control response to an EC₂₀ concentration of GABA by co-application with anaesthetic (where 0%=no effect). The amount of potentiation produced by an anaesthetic at a given mutated receptor was compared to the corresponding potentiation produced at the wild-type GABA_A α₂β₁ receptor. ^*, ^**, or ^*** indicates that the amount of potentiation at the mutated receptor was different from that at the wild-type GABA_A α₂β₁ receptor with a significance value of P<0.05, P<0.01, or P<0.005, respectively. Data for isoflurane, propofol (Krasowski et al., 1998b) and TCEt (Krasowski et al., 1998a) have been previously published.

Figure 8

Summary of the effects of mutations in TM2 or TM3 of the glycine α₁ receptor subunit on potentiation of submaximal glycine currents by (A) ethers, (B) primary alcohols along with the alkane trichloroethylene, and (C) tertiary alcohols. The ordinate depicts percentage change of a control response to an EC₂₀ concentration of glycine by co-application with anaesthetic. The amount of potentiation produced by an anaesthetic at a given mutated receptor was compared to the corresponding potentiation produced at the wild-type glycine α₁ receptor. ^*, ^**, or ^*** indicates that the amount of potentiation at the mutant receptor was different from that at the wild-type glycine α₁ receptor with a significance value of P<0.05, P<0.01, or P<0.005, respectively. Data for TCEt has been previously published (Krasowski et al., 1998a).