Modulation of α1β3γ2 GABAA receptors expressed in X. laevis oocytes using a propofol photoswitch tethered to the transmembrane helix Cecilia M. Borghesea,b,1, Hua-Yu L. Wangc, Stanton F. McHardyc, Robert O. Messinga,b, James R. Trudelld,e,2, R. Adron Harrisa,b, and Edward J. Bertaccinid,f aWaggoner Center for Alcohol and Addiction Research, The University of Texas at Austin, Austin, TX 78712; bDepartment of Neuroscience, The University of Texas at Austin, Austin, TX 78712; cCenter for Innovative Drug Discovery, University of Texas at San Antonio, San Antonio, TX 78249; dDepartment of Anesthesia, Stanford University, Palo Alto, CA 94305; eBeckman Program for Molecular and Genetic Medicine, Stanford University, Palo Alto, CA 94305; and fDepartment of Anesthesia, Palo Alto VA Health Care System, Palo Alto Division, Palo Alto, CA 94304 Edited by Ehud Y. Isacoff, University of California, Berkeley, CA, and approved January 13, 2021 (received for review April 29, 2020) Tethered photoswitches are molecules with two photo-dependent The GABAA receptor is formed by five subunits (usually two isomeric forms, each with different actions on their biological tar- α, two β, and one γ or δ) arranged in pseudosymmetry around a gets. They include reactive chemical groups capable of covalently central channel, in the following order (counterclockwise, viewed binding to their target. Our aim was to develop a β-subunit-teth- from the extracellular side): γ-β-α-β-α (2). Each interface is ered propofol photoswitch (MAP20), as a tool to better study the named after the subunits that form it, with “+” and “−” designated α β γ mechanism of anesthesia through the GABAA 1 3 2 receptor. We following the counterclockwise order (a model of the α1β3γ2 used short spacers between the tether (methanethiosulfonate), GABAA receptor can be found in SI Appendix,Fig.S1). Each the photosensitive moiety (azobenzene), and the ligand (propo- subunit consists of an extracellular domain, attached to a sequence fol), to allow a precise tethering adjacent to the putative propofol of four transmembrane helices (TMs), with a large intracellular + − binding site at the β α interface of the receptor transmembrane loop inserted between TM3 and TM4. Multiple anesthetic drugs helices (TMs). First, we used molecular modeling to identify possi- that act through this receptor possess relatively low binding affin- ble tethering sites in β3TM3 and α1TM1, and then introduced cys- ities; therefore, precise identification and characterization of their PHARMACOLOGY β teines in the candidate positions. Two mutant subunits [ 3(M283C) binding sites require the use of techniques like mutagenesis, α and 1(V227C)] showed photomodulation of GABA responses af- substituted cysteine modification protection (SCAMP), and pho- ter incubation with MAP20 and illumination with lights at specific tolabeling with photoreactive anesthetic analogs (3). Structures of wavelengths. The α1β3(M283C)γ2 receptor showed the greatest the GABAA receptor with bound anesthetic drugs have recently photomodulation, which decreased as GABA concentration in- been made available (4), but one unexplored way of obtaining creased. The location of the mutations that produced photomodu- valuable, functional information would be by using appropriately lation confirmed that the propofol binding site is located in the − designed tethered photoswitches. In previous studies of GABA β+α interface close to the extracellular side of the transmembrane A receptors, the photoswitches used have included diffusible propofol helices. Tethering the photoswitch to cysteines introduced in the photoswitches (5, 6) and a tethered propofol photoswitch with a positions homologous to β3M283 in two other subunits (α1W288 and γ2L298) also produced photomodulation, which was not en- tirely reversible, probably reflecting the different nature of each Significance interface. The results are in agreement with a binding site in the β+α− interface for the anesthetic propofol. Mechanisms of anesthesia remain obscure. We developed a β-subunit-specific tethered photoswitch containing the widely optopharmcology | anesthetic | molecular modeling | azobenzene | used anesthetic, propofol, to study the anesthetic mechanisms methanethiosulfonate through γ-aminobutyric acid type A (GABAA) receptors critical for this drug’s effects. Photoswitches are molecules that shift hile photoswitches have been a popular topic in numerous between two isomeric forms after irradiation with light at a Wreviews (1), their application in research is still very rare. specific wavelength, which would produce different biological Photoswitches are freely diffusible molecules containing a pho- outcomes on their targets. Tethered photoswitches possess tosensitive moiety which can alternate between two isomeric reactive groups that can covalently bind to their target. We forms depending on light irradiation at specific wavelengths. identified several residues in the receptor transmembrane do- These isomeric forms of the photoswitch possess different af- mains that are suitable for tethering. Once bound and after photoirradiation, the propofol moiety of MAP20 swings into its finities or efficacies toward their biological target, such that their binding site, allowing for light-controlled potentiation of the pharmacological activity can be turned on or off depending on GABA receptor expressed in Xenopus laevis oocytes. the light wavelength used, thus providing temporal and spatial A control. When photoswitches covalently tether to a native or Author contributions: C.M.B., J.R.T., R.A.H., and E.J.B. designed research; C.M.B., engineered residue at the specific biological target, the resulting H.-Y.L.W., S.F.M., J.R.T., and E.J.B. performed research; H.-Y.L.W. and S.F.M. contributed conformation would determine the corresponding pharmaco- new reagents/analytic tools; C.M.B., H.-Y.L.W., S.F.M., R.O.M., J.R.T., R.A.H., and E.J.B. logical activity. One way the tethered photoswitch can be acti- analyzed data; and C.M.B., H.-Y.L.W., S.F.M., R.O.M., R.A.H., and E.J.B. wrote the paper. vated is by positioning the tether in such a way that the ligand The authors declare no competing interest. moiety can reach its binding site in only one of the photoswitch This article is a PNAS Direct Submission. conformations. Additional major advantages of the tethered Published under the PNAS license. photoswitches are high local concentration (the residue cannot 1To whom correspondence may be addressed. Email: [email protected]. diffuse away) and spatial restriction within the biological target. 2Deceased July 29, 2019. The ligand groups of photoswitches can possess diverse phar- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ macological activities, acting as agonists, inverse agonists, or doi:10.1073/pnas.2008178118/-/DCSupplemental. antagonists at specific binding sites. Published February 15, 2021. PNAS 2021 Vol. 118 No. 8 e2008178118 https://doi.org/10.1073/pnas.2008178118 | 1of8 Downloaded by guest on September 30, 2021 very long spacer between the tethering cysteine (located in the this tethered photoswitch are connected by short spacers, decreas- extracellular domain) and the propofol moiety (6). Though all ing the range between tethering and target residues. We used β3 three positively modulated GABAA receptors, none could be used subunits in our study because the immobilizing and hypnotic effects to define the propofol binding site. of propofol are mostly mediated by β3-containing receptors (10). Multiple studies point to propofol binding cavities being lo- Results cated in the TM interfaces between GABAA receptor subunits. The observation that specific mutations in TM2 or TM3 of GABAA Before testing the photoswitch in relevant cysteine mutants, β subunits could dramatically decrease propofol potentiation of endogenous cysteines located in the TM and extracellular domains GABA responses, and even direct activation of the receptor (7–9) of the GABAA subunits were replaced by alanines, creating a led to the development of the β3(N265M) knockin mouse, which Cys-to-Ala background into which tethering cysteines could be showed either a greatly decreased or absent hypnotic effect of introduced without fear of endogenous cysteines interfering with propofol, depending on the test used (10). More recent studies have the interpretation of the photomodulation results. The function of focused on a more complete characterization of binding sites for the resulting Cys-to-Ala α1β3γ2GABAA receptor was compared propofol as well as other intravenous anesthetics. A photoreactive with the wild-type receptor, and no differences were observed, − propofol analog labeled three amino acids in the β+α interface except for a modest, albeit significant, increase in propofol po- − (β3M286, α1M236, and α1I239) and one in the α+β interface tentiation (SI Appendix, Fig. S2 and Table S1). (β3M227) in α1β3 receptors. Using etomidate and R-mTFD-MPAB We used molecular modeling to position propofol in the pu- β+α− [R-5-allyl-1-methyl-5-(m-trifluoromethyldiazirinylphenyl)barbituric tative binding site located in the interface in a GABAA acid] to inhibit photolabeling established that propofol also binds to receptor structure. This cavity is lined by several residues critical − the β+β interface, suggesting that propofol shows little selectivity for propofol action: βN265 and βM286, and αL232 and αM236. for either interface (11). Another study mutated photolabeled res- Then we added the