Identifying the Propofol Binding Site(S) in Heterologously Expressed Human Alpha1 Beta3 Gaba-A Receptors using a Photoreactive Propofol Analog

Abstract

Propofol, a widely used intravenous general anesthetic, potentiates GABA-A receptor (GABAAR) function at anesthetic concentrations, and “knock-in” mice expressing a single mutation at position 15 in the β3 subunit M2 helix (βM2-15) have reduced sensitivity to many of the anesthetic effects of propofol. However, the location of propofol's binding site(s) remains to be determined. Here we use a photoactive analog of propofol, [3H]m-azipropofol(2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol), an aryl diazirine with broad amino acid side chain reactivity, to identify propofol binding site(s) in affinity-purified, expressed human α1β3 GABAARs in detergent/asolectin solution. m-Azipropofol acts as a tadpole anesthetic with potency (EC50 = 3μM) similar to propofol, and it acts as a low efficacy GABAAR potentiator (Hall et al. 2010, J Med. Chem. 53, 5667). Irradiation of GABAAR for 30 min at 365nm resulted in [3H]m-azipropofol photoincorporation into both the α1 and β3 subunits that was inhibitable by propofol and non-radioactive m-azipropofol with IC50s of 28±3 μM and 6.7±1 μM, respectively. Individual amino acids photolabeled by [3H]m-azipropofol were identified by protein microsequencing of subunit fragments generated by enzymatic or chemical digestion. In β3, there was propofol-inhibitable photolabeling of β3Met-286, an amino acid that is photolabeled by [3H]azietomidate in the etomidate binding site at the β3M3-α1M1 interface (Li et al. 2006, J Neurosci. 26, 11599, Chiara et al. 2012, Biochem. 51, 836). In α1, [3H]m-azipropofol photolabeled α1Ile-239 within α1M1, located one helical turn below α1Met-236 that is photolabeled by [3H]azietomidate. Consistent with propofol and m-azipropofol binding to this etomidate site, they completely inhibit [3H]azietomidate photolabeling with IC50s of 46±3 μM and 23±3 μM, respectively. Both β3Met-286 and α1Met-236 are in close proximity to βM2-15, which docking calculations suggest may also be part of this propofol binding site.