Photo-activated azi-etomidate, a general anesthetic photolabel, irreversibly enhances gating and desensitization of gamma-aminobutyric acid type A receptors

Abstract

Background: The general anesthetic etomidate acts via gamma-aminobutyric acid type A (GABA(A)) receptors, enhancing activation at low GABA and prolonging deactivation. Azi-etomidate is a photo-reactive etomidate derivative with similar pharmacological actions, which has been used to identify putative binding sites. The authors examine the irreversible effects of azi-etomidate photo-modification on functional GABA(A) receptors in cell membranes.

Methods: GABA(A) receptors (alpha1beta2gamma2L) were expressed in both Xenopus oocytes and human embryonic kidney cells exposed to 365 nm light-activated azi-etomidate with or without GABA, then extensively washed. Receptor-mediated chloride currents were measured using voltage clamp electrophysiology to assess the ratio of peak responses at 10 microm and 1 mm GABA (I10/I1000) and deactivation time course.

Results: After azi-etomidate photo-modification, I10/I1000 ratios were persistently enhanced and deactivation was prolonged, mimicking reversible azi-etomidate actions. Azi-etomidate and ultraviolet light were required to produce irreversible receptor modulation. Adding GABA during photo-modification greatly enhanced irreversible modulation. Azi-etomidate modification also dose-dependently reduced maximal GABA-activated currents, consistent with accumulation of permanently desensitized receptors. Excess etomidate during azi-etomidate photo-modification competitively reduced permanent desensitization. Persistent channel modulation was blocked by 320-fold excess etomidate but enhanced when 32-fold excess etomidate was present.

Conclusions: Azi-etomidate efficiently photo-modifies etomidate sites on GABA(A) receptors in intact cells, producing persistent functional changes that mimic its reversible effects. The results demonstrate sequential modification at more than one etomidate site per receptor. The sites display reciprocal positive cooperativity. In combination with focal photo-activation, azi-etomidate may prove useful for studies of anesthetic actions in neural circuits.

Fig. 1

Reversible effects of azi-etomidate on α₁β₂γ_2L γ-aminobutyric acid type A (GABA_A) receptors expressed in human embryonic kidney cells. Three sets of traces are shown from a single voltage-clamped HEK293 cell, illustrating the relative peak amplitudes and deactivation kinetics of currents elicited with 10 μm or 1 mm GABA in the absence or presence of azi-etomidate. Bars above the traces indicate the timing of GABA (lower bars) and azi-etomidate (upper bar) application. (A) Traces were recorded before azi-etomidate exposure: I₁₀ = 198 pA; I₁₀₀₀ = 1020 pA; τ_w (weighted average time constant for deactivation) = 61 ms. (B) A single trace elicited with 10 μm GABA during exposure to 10 μm azi-etomidate: I₁₀ = 785 pA; τ_w = 950 ms. (C) Traces were recorded after azi-etomidate exposure and a 2-min wash: I₁₀ = 206 pA; I₁₀₀₀ = 1030 pA; τ_w = 78 ms. (D) Average normalized (to 1 mm GABA control) GABA-induced peak currents from HEK293 cells and patches in the absence (circles, four cells) and presence (squares, four cells) of 3.2 μm azi-etomidate. Fitted GABA EC₅₀ values in the absence and presence of azi-etomidate are, respectively, 43 ± 5.3 μm and 7.0 ± 0.54 μm. (E) 200 μm azi-etomidate was used to stimulate current from an excised outside-out membrane patch. Azi-etomidate application is indicated by the bar above the trace. The current desensitizes during azi-etomidate application with a time constant of 22 ± 0.4 s. The steady-state current after desensitization is 12% of peak.

Fig. 2

Irreversible effects of photo-activated azi-etomidate on γ-aminobutyric acid type A (GABA_A) receptors expressed in Xenopus oocytes and human embryonic kidney cells. (A) Average (±SD) ratio of currents elicited with 10 μm GABA(I₁₀) vs. 1 mm GABA (I₁₀₀₀) for six groups of oocytes expressing α₁β₂γ_2L receptors. The number of cells in each group is shown inside the bar, and factors included during a 5-min oocyte pretreatment are indicated below: ND96 is electrophysiology buffer; +UV = 365 nm light/–UV = room light; Azi-Eto = 3.2 μm azi-etomidate; GABA = 10 μm GABA. Oocytes were washed extensively (see “Materials and Methods”) after treatment. Only cells exposed to UV light, azi-etomidate, and GABA displayed significantly enhanced responses to low GABA. * Differs from control (ND96 –UV) at P < 0.05. (B–E) HEK 293 cells on coverslips were exposed to azi-etomidate and UV light in culture dishes, followed by extensive washing (see “Materials and Methods”). (B) GABA-activated currents were recorded from a cell after exposure to 365 nm light for 5 min. GABA concentration is indicated in micromolar, and the bar over the traces indicates GABA application. I₁₀/I₁₀₀₀ = 0.12; deactivation τ_w (weighted average time constant for deactivation) = 50 ms. (C) GABA-activated currents were recorded from a cell after exposure to 10 μm azi-etomidate + 10 μm GABA + 365 nm light for 5 min, then washed for 10 min. I₁₀/I₁₀₀₀ = 0.52; deactivation τ_w = 320 ms. (D) Average normalized (to 1 mm GABA control) GABA-induced peak currents from HEK293 cells and patches after no treatment (circles, five cells; EC₅₀ = 46 ± 4.4 μm) or photo-modification with 3.2 μm azi-etomidate + 10 μm GABA + 365 nm light for 5 min, then washed for 10 min (triangles, six cells; EC₅₀ = 17 ± 6.3 μm). (E) I₁₀/I₁₀₀₀ data from HEK293 cells after various 5-min treatments and subsequent washing. Treatment conditions are indicated below each bar, and the number of cells is indicted in each bar: ECF = electrophysiology buffer; UV = 365 nm light; Azi = 3.2 μm azi-etomidate; GABA = 10 μm GABA; Eto = 100 μm etomidate. * Differs from ECF control at P < 0.05. ** Differs both from ECF control and from Azi + GABA + UV at P < 0.01.

Fig. 3

Irreversible effects of photo-activated azi-etomidate on γ-aminobutyric acid type A (GABA_A) receptors expressed in a single human embryonic kidney cell. (A–D) Current traces recorded from a single HEK293 cell expressing GABA_A receptors. (A) GABA currents recorded before treatment. GABA concentration is labeled in micromolar, and the bar over the traces indicates drug application. The ratio of peak currents at 10 μm versus 1 mm GABA, I₁₀/I₁₀₀₀ = 0.16; Deactivation τ_w (weighted average time constant) = 60 ms. (B) GABA currents recorded after 5 min cell treatment (on the microscope stage) with 3.2 μm azi-etomidate + 10 μm GABA + 365 nm light followed by 10-min wash. I₁₀/I₁₀₀₀ = 0.29; Deactivation τ_w = 590 ms. Maximal GABA current (I₁₀₀₀) is significantly reduced after treatment (I_post/I_pre = 0.42). (C) Current elicited with 100 μm propofol before treatment. The maximal current normalized to the 1 mm GABA current in A is 0.41. (D) Current elicited with 100 μm propofol after treatment with azi-etomidate. The maximal current normalized to the 1 mm GABA current in B is 0.85. (E) Summary of I₁₀/I₁₀₀₀ results from cells treated on the microscope stage. Treatment conditions are indicated below the bars (as given in fig. 2, and azi-etomidate concentrations are indicated in micro-molar) and number of cells inside the bars. Deactivation τ_w data show similar effects. * Differs from ECF control at P < 0.05.

Fig. 4

Wash after azi-etomidate modification: reversible vs. irreversible effects. (A and B) Currents from a single HEK293 cell both before treatment (A: premodification) and during wash after treatment with 1 μm azi-etomidate + 10 μm GABA + 365 nm light. Currents were elicited with 1 mm γ-aminobutyric acid (GABA). Bars over traces indicate GABA application, and traces are labeled with wash time in minutes (the 6-min washout trace is unlabeled for clarity). (C) Peak current amplitude from Panels A and B is plotted vs. wash time. For this cell, recovery is 90% complete in 6 min and I_post/I_pre = 0.85. (D) Deactivation τ_w (weighted average time constant) is plotted vs. wash time. Deactivation continues to slow during wash (E) Maximal currents after treatment and wash normalized to pretreatment currents vs. azi-etomidate. Cells were treated with azi-etomidate + 10 μm GABA + 365 nm light for 5 min and washed for at least 10 min. Average I_post/I_pre ± SD results are shown from at least three cells or patches at each azi-etomidate concentration. (F) Washout time vs. azi-etomidate. Cells were treated as described for Panel E and time to 90% recovery of maximal amplitude (I₁₀₀₀) or deactivation weighted time constant (τ_w) was recorded. Whereas the averaged data show a trend toward slower washout after treatment with higher azi-etomidate concentrations, there were no significant differences among groups.

Fig. 5

Excess etomidate during azi-etomidate photo-modification blocks irreversible effects. (A and B) Currents elicited with 1 mm GABA from a single excised outside-out patch. The GABA application period is indicated by the bar above the trace. (A) Current before treatment. Deactivation τ_w = 105 ms. (B) Current recorded after treatment for 5 min with 3.2 μm azi-etomidate + 100 μm etomidate + 10 μm GABA + 365 nm light and wash for 15 min. I_post/I_pre = 0.98 and deactivation τ_w = 390 ms. Maximal current is not reduced, but deactivation remains prolonged. (C–E) Examples of deactivation current traces from individual patches subjected to photo-modification using 0.32 μm azi-etomidate and GABA with or without 320-fold excess (100 μm) etomidate. (C) Deactivation current before photo-modification; τ_w = 90 ms. (D) Deactivation current after photo-modification in the presence of 320-fold excess etomidate (same patch as C); τ_w = 97 ms. (E) Deactivation current after photo-modification without excess etomidate; τ_w = 330 ms. (F) Summary of competition studies at 0.32 μm azi-etomidate. Bars represent deactivation τ_w data (mean ± SD) with the number of patches indicated. * Differs from control at P < 0.05.