Channel opening by anesthetics and GABA induces similar changes in the GABAA receptor M2 segment

Abstract

For many general anesthetics, their molecular basis of action involves interactions with GABA(A) receptors. Anesthetics produce concentration-dependent effects on GABA(A) receptors. Low concentrations potentiate submaximal GABA-induced currents. Higher concentrations directly activate the receptors. Functional effects of anesthetics have been characterized, but little is known about the conformational changes they induce. We probed anesthetic-induced conformational changes in the M2 membrane-spanning, channel-lining segment using disulfide trapping between engineered cysteines. Previously, we showed that oxidation by copper phenanthroline in the presence of GABA of the M2 6' cysteine mutants, alpha(1)T261Cbeta(1)T256C and alpha(1)beta(1)T256C resulted in formation of an intersubunit disulfide bond between the adjacent beta-subunits that significantly increased the channels' spontaneous open probability. Oxidation in GABA's absence had no effect. We examined the effect on alpha(1)T261Cbeta(1)T256C and on alpha(1)beta(1)T256C of oxidation by copper phenanthroline in the presence of potentiating and directly activating concentrations of the general anesthetics propofol, pentobarbital, and isoflurane. Oxidation in the presence of potentiating concentration of anesthetics had little effect. Oxidation in the presence of directly activating anesthetic concentrations significantly increased the channels' spontaneous open probability. We infer that activation by anesthetics and GABA induces a similar conformational change at the M2 segment 6' position that is related to channel opening.

FIGURE 1

Aligned channel-lining residues in the α₁ and β₁ M2 membrane-spanning segments. Position of the 6′ residues is highlighted in reverse contrast. Index numbers are shown in the center to facilitate comparisons with other members of the gene superfamily (27). Solid squares indicate channel-lining positions, solid circles non-channel-lining positions based on SCAM experiments (25). Zn indicates the Zn²⁺ binding site location at β₁His267 (18), and PTX indicates the picrotoxin binding site location at the 2′ level (41).

FIGURE 2

Effect of Cu:phen-induced oxidation in the presence of GABA on single-channel currents recorded from α₁β₁T256C-containing receptors. (A and B) All-points histogram of currents recorded from an HEK 293 cell in the cell-attached configuration (left) and the corresponding current recordings (right). The pipette did not contain GABA. The pipette voltage was clamped to +60 mV (hyperpolarization of the cell). Data traces were numerically filtered to 1 kHz for display using QuB Software (59). The scale bars represent 2 pA and 50 ms. (A) Cell-attached patch recording from a cell expressing α₁β₁T256C receptors. No GABA_A receptor channel activity was observed. The current amplitude histogram was fitted by a Gaussian distribution centered around −0.05 ± 0.1 pA. (B) Cell-attached patch recording from an α₁β₁T256C-transfected cell that was treated with Cu:phen in the presence of 5 μM GABA for 10–15 min. Before patch formation, GABA and Cu:phen were thoroughly washed out of the dish. After oxidation in the presence of GABA, a significant amount of single-channel activity was observed with no GABA in the pipette. The current amplitudes were best fitted using the sum of two Gaussian distributions centered around −0.06 ± 0.22 pA, relative area = 82%, and 1.19 ± 0.34 pA, relative area = 18%.

FIGURE 3

Anesthetic concentration-response relationships for potentiation and direct activation. (A) Current traces from an oocyte expressing α₁T261Cβ₁T256C receptors. Periods of GABA and propofol application are indicated by the black bars above the current traces. Propofol concentration (μM) is indicated above bars. GABA concentration was 0.4 μM except for the second trace, where it was 20 μM. Traces are separated by 5-min washout periods. (B) Pentobarbital direct activation and potentiation of GABA currents from an oocyte expressing α₁T261Cβ₁T256C receptors. Periods of GABA and pentobarbital application are indicated by the black bars above the current traces. Pentobarbital concentration (μM) is indicated above bars. The prominent rebound currents seen after pentobarbital and GABA washout, particularly in the 300 and 1000 μM pentobarbital applications, represent relief of inhibition by pentobarbital. Traces are separated by 5-min washout periods. (C) Potentiating isoflurane concentration responses on current traces from an oocyte expressing α₁β₁T256C receptors. Isoflurane concentration (μM) above the bars. GABA concentration was 0.5 μM. (D) Direct activation and inhibition by isoflurane. Current traces from an oocyte expressing α₁β₁T256C receptors. Isoflurane concentration was 20 mM, GABA concentration was 5 μM.

FIGURE 4

Effect of Cu:phen-induced oxidation in the presence of potentiating concentrations of propofol and pentobarbital on currents from oocytes expressing α₁T261Cβ₁T256C receptors. Dotted line indicates the initial holding current level. Note that the resting holding currents indicated by the initial currents at the start of the traces after Cu:phen application are similar to the initial holding currents before Cu:phen application. Periods of reagent application are indicated by the black bars above the current traces. (A) Oxidation in the presence of a potentiating concentration of propofol, 2 μM. (B) Oxidation in the presence of a potentiating concentration of pentobarbital, 30 μM.

FIGURE 5

Effect of Cu:phen-induced oxidation in the presence of directly activating concentrations of the three anesthetics. Top dotted line indicates the initial holding current level. Lower dotted line indicates level of holding current after Cu:phen application. Note the increase in holding currents indicated by the arrows (between the dotted lines and indicated as holding current). Also note the decrease in the subsequent GABA-induced currents after application of Cu:phen. Periods of reagent applications are indicated by the black bars above the current traces. (A) Oxidation in the presence of an activating concentration of propofol, 40 μM. Oocyte expressing α₁T261Cβ₁T256C receptors. (B) Oxidation in the presence of an activating concentration of pentobarbital, 1 mM. Oocyte expressing α₁T261Cβ₁T256C receptors. (C) Oxidation in the presence of an activating concentration of isoflurane, 20 mM. At this isoflurane concentration there is also a significant amount of inhibition by isoflurane. Oocyte expressing α₁β₁T256C receptors.