Tryptophan mutations at azi-etomidate photo-incorporation sites on alpha1 or beta2 subunits enhance GABAA receptor gating and reduce etomidate modulation

Abstract

The potent general anesthetic etomidate produces its effects by enhancing GABA(A) receptor activation. Its photolabel analog [(3)H]azi-etomidate labels residues within transmembrane domains on alpha and beta subunits: alphaMet236 and betaMet286. We hypothesized that these methionines contribute to etomidate sites formed at alpha-beta subunit interfaces and that increasing side-chain bulk and hydrophobicity at either locus would mimic etomidate binding and block etomidate effects. Channel activity was electrophysiologically quantified in alpha(1)beta(2)gamma(2L) receptors with alpha(1)M236W or beta(2)M286W mutations, in both the absence and the presence of etomidate. Measurements included spontaneous activation, GABA EC(50), etomidate agonist potentiation, etomidate direct activation, and rapid macrocurrent kinetics. Both alpha(1)M236W and beta(2)M286W mutations induced spontaneous channel opening, lowered GABA EC(50), increased maximal GABA efficacy, and slowed current deactivation, mimicking effects of etomidate on alpha(1)beta(2)gamma(2L) channels. These changes were larger with alpha(1)M236W than with beta(2)M286W. Etomidate (3.2 muM) reduced GABA EC(50) much less in alpha(1)M236Wbeta(2)gamma(2L) receptors (2-fold) than in wild type (23-fold). However, etomidate was more potent and efficacious in directly activating alpha(1)M236Wbeta(2)gamma(2L) compared with wild type. In alpha(1)beta(2)M286Wgamma(2L) receptors, etomidate induced neither agonist-potentiation nor direct channel activation. These results support the hypothesis that alpha(1)Met236 and beta(2)Met286 are within etomidate sites that allosterically link to channel gating. Although alpha(1)M236W produced the larger impact on channel gating, beta(2)M286W produced more profound changes in etomidate sensitivity, suggesting a dominant role in drug binding. Furthermore, quantitative mechanistic analysis demonstrated that wild-type and mutant results are consistent with the presence of only one class of etomidate sites mediating both agonist potentiation and direct activation.

Figure 1. GABA Concentration-Responses in the Absence and Presence of Etomidate

Data points represent mean ± sd (n ≥ 8) peak oocyte currents normalized to maximal GABA-elicited currents in the absence of etomidate. Open symbols represent control conditions and solid symbols represent experiments in the presence of 3.2 μM etomidate. Lines represent logistic (Eq. 1, Methods) fits to data in the absence (solid) and presence (dashed) of etomidate. GABA EC₅₀ ratios (control/3.2 μM ETO) are reported in Table 1. A: Wild-type α₁β₂γ_2L receptors: Control (open circles): A = 1.02 ± 0.01; EC₅₀ = 43 ± 1.7 μM; nH = 1.3 ± 0.12. 3.2 μM Eto (solid circles): A = 1.17 ± 0.02; EC₅₀ = 1.9 ± 0.12 μM; nH = 1.5 ± 0.13. B: α₁M236Wβ₂γ_2L receptors: Control (open squares): A = 1.00 ± 0.013; EC₅₀ = 2.0 ± 0.10 μM; nH = 1.2 ± 0.11. 3.2 μM Eto (solid squares): A = 0.98 ± 0.023; EC₅₀ = 1.2 ± 0.18 μM; nH = 1.4 ± 0.25. C: α₁β₂M286Wγ_2L receptors: Control (open diamonds): A = 1.06 ± 0.07; EC₅₀ = 6.6 ± 1.3 μM; nH = 1.2 ± 0.34. 3.2 μM Eto (solid diamonds): A = 1.09 ± 0.11; EC₅₀ = 6.2 ± 2.1 μM; nH = 1.3 ± 0.36.

Figure 2. Etomidate Direct Activation Concentration-Responses

Data points represent mean ± sd (n > 5) peak oocyte currents normalized to maximal GABA-elicited currents. Lines represent logistic (Eq. 1, Methods) fits to data. Wild-type α₁β₂γ_2L receptors (circles): A = 0.39 ± 0.062; EC₅₀ = 31 ± 12 μM; nH = 1.3 ± 0.23. α₁M236Wβ₂γ_2L receptors (squares): A = 0.97 ± 0.072; EC₅₀ = 12 ± 2.7 μM; nH = 1.5 ± 0.21. α₁β₂M286Wγ_2L receptors (diamonds): No fit.

Figure 3. Estimation of spontaneous activation and maximal GABA efficacy

Sweeps were recorded from oocytes expressing receptors as labeled. Top panels show examples of current recordings illustrating responses to 2 mM PTX and 10 mM GABA. Wild-type receptors display no detectable PTX-sensitive spontaneous leak current. α₁M236Wβ₂γ_2L and α₁β₂M286Wγ_2L receptors both display outward currents, representing closure of spontaneously open channels. Results are summarized in Table 1. Lower panels show examples of current recordings during multi-solution experiments designed to estimate maximal GABA gating efficacy. Currents were initially elicited with 10 mM GABA (I_GABA), and 2 μM alphaxalone was then added after the maximal GABA response was observed (I_GABA+Alphax). Note that alphaxalone enhances wild-type currents by about 20%, and much less enhancement (1% or less) is seen in currents elicited from the mutant channels. Estimated GABA efficacies are summarized in Table 1.

Figure 4. Activation, desensitization and deactivation kinetics

Each panel shows a current trace recorded from an HEK293 patch subjected to a 1.0 s GABA pulse (1-3 mM). Black bars over traces represent GABA application period. A: Wild type α₁β₂γ_2L receptors. B: α₁M236Wβ₂γ_2L receptors. C: α₁β₂M286Wγ_2L receptors. Current activation and desensitization rates are similar for all three traces, while deactivation of both mutants is significantly slower than wild-type. Average time constants results are reported in Table 2.

Figure 5. Monod-Wyman-Changeux Co-Agonist Models for GABA and Etomidate Concentration-Responses

Average data from figures 1 and 2 (symbols) was transformed into estimated P_open values using equation 2 in Methods. Equation 3 (Methods) was globally fitted to combined P_open data for each channel with both [GABA] and [ETO] as free parameters. Fitted models are represented by lines through the data points. Solid lines and open symbols represent control GABA responses. Dashed lines and solid symbols represent GABA responses in the presence of 3.2 μM etomidate. Dash-dotted lines and crossed symbols represent etomidate direct activation. A: Wild type α₁β₂γ_2L receptors. B: α₁M236Wβ₂γ_2L receptors. C: α₁β₂M286Wγ_2L receptors. Fitted parameters are reported in Table 3.