Mutations at Beta N265 in γ-Aminobutyric Acid Type A Receptors Alter Both Binding Affinity and Efficacy of Potent Anesthetics

<div><p>Etomidate and propofol are potent general anesthetics that act via GABAA receptor allosteric co-agonist sites located at transmembrane β+/α− inter-subunit interfaces. Early experiments in heteromeric receptors identified βN265 (M2-15′) on β2 and β3 subunits as an important determinant of sensitivity to these drugs. Mechanistic analyses suggest that substitution with serine, the β1 residue at this position, primarily reduces etomidate efficacy, while mutation to methionine eliminates etomidate sensitivity and might prevent drug binding. However, the βN265 residue has not been photolabeled with analogs of either etomidate or propofol. Furthermore, substituted cysteine modification studies find no propofol protection at this locus, while etomidate protection has not been tested. Thus, evidence of contact between βN265 and potent anesthetics is lacking and it remains uncertain how mutations alter drug sensitivity. In the current study, we first applied heterologous α1β2N265Cγ2L receptor expression in Xenopus oocytes, thiol-specific aqueous probe modification, and voltage-clamp electrophysiology to test whether etomidate inhibits probe reactions at the β-265 sidechain. Using up to 300 µM etomidate, we found both an absence of etomidate effects on α1β2N265Cγ2L receptor activity and no inhibition of thiol modification. To gain further insight into anesthetic insensitive βN265M mutants, we applied indirect structure-function strategies, exploiting second mutations in α1β2/3γ2L GABAA receptors. Using α1M236C as a modifiable and anesthetic-protectable site occupancy reporter in β+/α− interfaces, we found that βN265M reduced apparent anesthetic affinity for receptors in both resting and GABA-activated states. βN265M also impaired the transduction of gating effects associated with α1M236W, a mutation that mimics β+/α− anesthetic site occupancy. Our results show that βN265M mutations dramatically reduce the efficacy/transduction of anesthetics bound in β+/α− sites, and also significantly reduce anesthetic affinity for resting state receptors. These findings are consistent with a role for βN265 in anesthetic binding within the β+/α− transmembrane sites.</p></div

Modification and protection at α1M236C reflects anesthetic site occupancy.

<p><b>Panel A)</b> Data represent mean ± SD peak current responses to GABA from oocytes (n = 4) expressing β3-α1M236C/β3-α1M236C-γ2L GABA_A receptors, normalized to maximal GABA responses. Lines represent logistic fits to responses using GABA alone (solid circles; EC₅₀ = 58 µM) and GABA with 3.2 µM etomidate (open circles; EC₅₀ = 3.2 µM). <b>Panel B)</b> Data represent mean ± SD peak current responses to etomidate from oocytes (n = 3) expressing β3-α1M236C/β3-α1M236C-γ2L GABA_A receptors, normalized to maximal GABA responses. The line represents a logistic fit with etomidate EC₅₀ = 47 µM. <b>Panel C)</b> A single voltage-clamp current trace illustrating maximal GABA (3 mM; white bar above trace) efficacy in oocyte-expressed β3-α1M236C/β3-α1M236C-γ2L GABA_A receptors, enhanced with addition of 10 µM etomidate (black bar). <b>Panel D)</b> Data from panels A and B were renormalized to maximal GABA efficacy (methods; Eq. 2) and fitted with a global MWC equilibrium co-agonist equation (methods; Eq. 3). Lines through data points represents the fitted MWC model: L₀ = 10,000; K_G = 42±8.7 µM; c = 0.0075±0.00048; K_E = 50±12 µM; d = 0.0089±0.00096. <b>Panel E)</b> Traces are from a single oocyte expressing β3-α1M236C/β3-α1M236C-γ2L GABA_A receptors, demonstrating the effects of repeated pCMBS applications on the relative responses to low versus high GABA stimulation. <b>Panel F)</b> Points represent response ratios to low (EC10) vs. high GABA, normalized to pre-modification control values. Lines through data represent linear fits used to determine relative bimolecular modification rates: GABA+pCMBS (circles; 1200±57 M⁻¹s⁻¹); GABA+pCMBS +5 µM alphaxalone (triangles; 3700±270 M⁻¹s⁻¹); and GABA+pCMBS +32 µM etomidate (squares; 106±8.4 M⁻¹s⁻¹). <b>Panel G)</b> Summary of modification rate results (mean ± se) for all oocytes expressing β3-α1M236C/β3-α1M236C-γ2L GABA_A receptors under different conditions. The rate with pCMBS alone is significantly accelerated with addition of GABA and GABA/alphaxalone. Relative to GABA+alphaxalone, modification in the presence of GABA is slowed 65% by 3 µM etomidate, 95% by 32 µM etomidate, and 91% by 30 µM propofol. * p<0.05; ** p<0.01.</p

The βN265M mutation reduces the channel gating effects of a α1M236W mutation that mimics etomidate.

<p><b>Panel A)</b> Data are reproduced from Guitchounts et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111470#pone.0111470-Guitchounts1" target="_blank">[38]</a> showing mean ± SD GABA concentration-response, normalized to maximal currents, from oocytes expressing concatenated receptor dimer and trimer constructs. Lines through data points represent non-linear least squares logistic fits (Eq. 1, methods). Solid squares represent β2-α1/γ2L-β2-α1 receptors (GABA EC₅₀ = 36 µM) and open circles represent β2-α1M236W/γ2L-β2-α1M236W receptors (EC₅₀ = 1.0 µM). <b>Panel</b> <b>B)</b> Data points are mean ± SD current responses to GABA, normalized to maximal currents. Solid diamonds represent β2N265M-α1/γ2L-β2N265M-α1 receptors (GABA EC₅₀ = 76 µM; 95% CI = 63 to 92 µM) and open triangles represent β2N265M-α1M236W/γ2L-β2N265M-α1M236W- receptors (EC₅₀ = 25 µM; 95% CI = 22 to 29 µM).</p

Wild-type and Mutant GABA_A receptor functional characteristics in <i>Xenopus</i> oocytes.

<p>Wild-type and Mutant GABA_A receptor functional characteristics in <i>Xenopus</i> oocytes.</p

Effects of βN265M mutation on etomidate in inactive versus active GABA_A receptors.

<p>Monod-Wyman-Changeux equilibrium allosteric models are diagrammed for both α1M236Cβ2γ2L (A) and α1M236Cβ2N265Mγ2L (B) receptors, each with two equivalent etomidate sites. Etomidate dissociation constants for both inactive (R; K_E) and GABA-activated (O; dK_E) receptors are rounded estimates based on both functional analysis and modification/protection results (Figs. 6 and 7). Etomidate-sensitive α1M236Cβ2γ2L receptors bind etomidate 100-fold more avidly in the active vs. inactive state, resulting in a 10,000-fold shift in the open-closed equilibrium constant (d²L₀ vs. L₀) when both sites are occupied. In contrast, etomidate-insensitive α1M236Cβ2N265Mγ2L receptors display low affinity (K_E ≈ dK_E ≈ 300 µM) for etomidate in both resting and GABA-activated states. Thus, high etomidate concentrations result in only partial site occupancy and weak modulatory effects.</p