Modulation of α1β3γ2 GABAA receptors expressed in X. laevis oocytes using a propofol photoswitch tethered to the transmembrane helix

Abstract

Tethered photoswitches are molecules with two photo-dependent isomeric forms, each with different actions on their biological targets. They include reactive chemical groups capable of covalently binding to their target. Our aim was to develop a β-subunit-tethered propofol photoswitch (MAP20), as a tool to better study the mechanism of anesthesia through the GABA_A α1β3γ2 receptor. We used short spacers between the tether (methanethiosulfonate), the photosensitive moiety (azobenzene), and the ligand (propofol), to allow a precise tethering adjacent to the putative propofol binding site at the β⁺α^- interface of the receptor transmembrane helices (TMs). First, we used molecular modeling to identify possible tethering sites in β3TM3 and α1TM1, and then introduced cysteines in the candidate positions. Two mutant subunits [β3(M283C) and α1(V227C)] showed photomodulation of GABA responses after incubation with MAP20 and illumination with lights at specific wavelengths. The α1β3(M283C)γ2 receptor showed the greatest photomodulation, which decreased as GABA concentration increased. The location of the mutations that produced photomodulation confirmed that the propofol binding site is located in the β⁺α^- interface close to the extracellular side of the transmembrane helices. Tethering the photoswitch to cysteines introduced in the positions homologous to β3M283 in two other subunits (α1W288 and γ2L298) also produced photomodulation, which was not entirely reversible, probably reflecting the different nature of each interface. The results are in agreement with a binding site in the β⁺α^- interface for the anesthetic propofol.

Keywords: anesthetic; azobenzene; methanethiosulfonate; molecular modeling; optopharmcology.

Fig. 1.

Tethered photoswitch. (A) Methanethiosulfonate azobenzene propofol 2020 (MAP20) consists of propofol (ligand group), an azobenzene moiety (photosensitive), and a tether (methanethiosulfonate). (B) The azobenzene moiety can change between cis and trans isomeric forms depending on the wavelength of the light irradiated.

Fig. 2.

GABA_A receptors containing β3(M283C) or α1(V227C) subunits. (A–D) Models of the β⁺α⁻ interface (β in light gray, α in dark gray), with MAP20 attached to β3(M283C), depicted in blue (A and B) and α1(V227C), depicted in cyan (C and D). Propofol moiety from MAP20 in purple, residues lining the putative binding site of propofol at this interface in green. Side view of the interface as seen from the membrane in A and C. View of the interface from the extracellular side in B and D. The relative position of the models is indicated by the rotation axis and degree symbol. (E and H) Photopotentiation of submaximal GABA responses in α1β3(M283C)γ2 and α1(V227C)β3γ2 GABA_A receptors. Representative tracings of photomodulation in (E) α1β3γ2 Cys-to-Ala, (F) α1β3(M283C)γ2, and (G) α1(V227C)β3γ2. Light wavelength: G (green), 520 nm; V (violet), 380 nm. (H) Quantitative summary of photomodulation in these receptors. Data are shown as mean ± SEM and were analyzed using one-way ANOVA, followed by Dunnett’s multiple comparisons test, *P < 0.05, **P < 0.01 versus Cys-to-Ala receptor (n = 4 to 5).

Fig. 3.

Photomodulation at different GABA concentrations after incubation with 50 µM MAP20. Representative tracings of (A) α1β3γ2 Cys-to-Ala and (B) α1β3(M283C)γ2 GABA_A receptors. (C) Quantitative summary of the photomodulation at different GABA concentrations. Data are shown as mean ± SEM, error bars not shown are smaller than symbols. Data were analyzed using two-way repeated measures ANOVA, followed by Dunnett’s multiple comparisons test, ***P < 0.01 versus 0.3 µM concentration (n = 4 to 5).

Fig. 4.

GABA_A receptors containing α1(W288C) or γ2(L298C) subunits. (A and B) Model of the α⁺β⁻ interface (α in dark gray, β in light gray), with MAP20 attached to α1(W288C) (in cyan). (C and D) Model of the γ⁺α⁻ interface (γ in gold, α in dark gray), with MAP20 attached to γ2(L298C) (in cyan). Propofol moiety from MAP20 in purple, residues lining the putative binding site of propofol at this interface in green. Side view of the interface as seen from the membrane in A and C. View of the interface from the extracellular side in B and D. The relative position of the models is indicated by the rotation axis and degree symbol. (E–G) Photopotentiation of submaximal GABA responses in α1(W288C)β3γ2 and α1β3γ2(L298C) GABA_A receptors. Representative tracings of photomodulation in (E) α1(W288C)β3γ2 and (F) α1β3γ2(L298C). Light wavelength: G (green), 520 nm; V (violet), 380 nm. (G) Quantitative summary of photomodulation in these mutants. Data are shown as mean ± SEM and analyzed using one-way ANOVA, followed by Dunnett’s multiple comparisons test, *P < 0.05 versus Cys-to-Ala, (n = 4 to 8).

Fig. 5.

Diagram of the GABA_A receptor transmembrane domains viewed from the extracellular side. MAP20 tethered to (A) β subunits, (B) α subunits, and (C) γ subunit. Orange circles mark the interfaces where putative binding sites for propofol exist (four binding sites, the α⁺γ⁻ interface is apparently lacking a propofol binding site). MAP20 is shown as a black and purple sphere-shaped molecule, with purple representing the propofol moiety.