Etomidate, propofol and the neurosteroid THDOC increase the GABA efficacy of recombinant alpha4beta3delta and alpha4beta3 GABA A receptors expressed in HEK cells

Abstract

General anesthetics, once thought to exert their effects through non-specific membrane effects, have highly specific ion channel targets that can silence neuronal populations in the nervous system, thereby causing unconsciousness and immobility, characteristic of general anesthesia. Inhibitory GABA(A) receptors (GABA(A)Rs), particularly highly GABA-sensitive extrasynaptic receptor subtypes that give rise to sustained inhibitory currents, are uniquely sensitive to GABA(A)R-active anesthetics. A prominent population of extrasynaptic GABA(A)Rs is made up of alpha4, beta2 or beta3, and delta subunits. Considering the demonstrated importance of GABA receptor beta3 subunits for in vivo anesthetic effects of etomidate and propofol, we decided to investigate the effects of GABA anesthetics on "extrasynaptic" alpha4beta3delta and also binary alpha4beta3 receptors expressed in human embryonic kidney (HEK) cells. Consistent with previous work on similar receptor subtypes we show that maximal GABA currents through "extrasynaptic" alpha4beta3delta receptors, receptors defined by sensitivity to EtOH (30mM) and the beta-carboline beta-CCE (1microM), are enhanced by the GABA(A)R-active anesthetics etomidate, propofol, and the neurosteroid anesthetic THDOC. Furthermore, we show that receptors formed by alpha4beta3 subunits alone also show high GABA sensitivity and that saturating GABA responses of alpha4beta3 receptors are increased to the same extent by etomidate, propofol, and THDOC as are alpha4beta3delta receptors. Therefore, both alpha4beta3 and alpha4beta3delta receptors show low GABA efficacy, and GABA is also a partial agonist on certain binary alphabeta receptor subtypes. Increasing GABA efficacy on alpha4/6beta3delta and alpha4beta3 receptors is likely to make an important contribution to the anesthetic effects of etomidate, propofol and the neurosteroid THDOC.

Fig. 1. Expression of the δ subunit with α4 and β3 leads to increased GABA-sensitivity

A,B) Representative recordings showing GABA dose response data from cells transfected with (A) α4β3 and (B) α4β3δ receptors. GABA concentrations of 0.1, 0.3, 1, 3, 10 and 30 µM were applied. The 0.3 µM GABA responses are marked below the traces; C) Average GABA dose response curve of α4β3δ and α4β3 receptors. GABA EC₅₀ values ± SD are, α4β3 = 1.3 ± 0.1 µM (n=8), for α4β3δ EC₅₀ = 0.53 ± 0.02 µM (n=6). The GABA sensitivities of receptors made from α4β3 and α4β3δ subunits are significantly different (p<0.01).

Fig. 2. Expression of the δ subunit with α4β3 confers sensitivity to 30 mM EtOH, 1 µM β-CCE and insensitivity to 1 µM Zn²⁺

Shown are representative recordings from three (α4β3) or four (α4β3δ) similar recordings. To evoke currents, 300 nM GABA was applied, either alone (black trace), with 30 mM EtOH (green trace), with 1 µM β-CCE (pink), or with 1 µM Zn²⁺ (light blue).

Fig. 3. Both α4β3 and α4β3δ receptors are enhanced by the anesthetics THDOC, etomidate and propofol at saturating (100 µM) GABA concentrations

A bar graph summarizing the mean enhancement relative to control for 3 µM etomidate, 3 µM propofol and 1 µM THDOC. Open bars show effects on α4β3 (n=9 for etomidate and propofol, n=8 for THDOC), shaded bars on α4β3δ (n=3). Enhancements of 100 µM GABA evoked peak current responses by 3 µM etomidate and propofol, and 1 µM THDOC are statistically significant (p < 0.01). A comparison of αβ3δ vs. αβ receptors responses to THDOC, etomidate and propofol shows that differences are not statistically significant (p=0.32 for etomidate, p=0.76 for propofol, p=0.69 for THDOC).

Fig. 4. In mutant α4β3N265M binary receptors the enhancing effects of 3 µM etomidate and 3 µM propofol but not 1 µM THDOC are abolished

The responses shown in A, B and C are from the same recording from a single α4β3N265M transfected cell (out of four similar recordings). (A) Etomidate (3 µM), (B) propofol (3 µM) and (C) THDOC (1 µM) were applied either alone or together with a saturating (100 µM) GABA concentration and compared to a saturating 100 µM GABA response. (D) Summary data (n=4, error bars are standard error of mean). Voltage clamp errors occurring with larger GABA currents may lead to underestimates in the amounts of current enhancement by anesthetics and overestimates of the amount of variability, particularly in the case of THDOC.