Multiple Propofol-binding Sites in a γ-Aminobutyric Acid Type A Receptor (GABAAR) Identified Using a Photoreactive Propofol Analog

Background: Propofol binding to GABA(A)R sites of uncertain location potentiates receptor function and produces anesthesia in vivo. Results: A photoreactive propofol analog identifies propofol-binding sites in 13 GABA(A)Rs. Conclusion: Propofol binds to each class of intersubunit sites in the GABA(A)R transmembrane domain. Significance: This study demonstrates that propofol binds to the same sites in a GABA(A)R as etomidate and barbiturates. Propofol acts as a positive allosteric modulator of -aminobutyric acid type A receptors (GABA(A)Rs), an interaction necessary for its anesthetic potency in vivo as a general anesthetic. Identifying the location of propofol-binding sites is necessary to understand its mechanism of GABA(A)R modulation. [H-3]2-(3-Methyl-3H-diaziren-3-yl)ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate (azietomidate) and R-[H-3]5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (mTFD-MPAB), photoreactive analogs of 2-ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate (etomidate) and mephobarbital, respectively, have identified two homologous but pharmacologically distinct classes of intersubunit-binding sites for general anesthetics in the GABA(A)R transmembrane domain. Here, we use a photoreactive analog of propofol (2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol ([H-3]AziPm)) to identify propofol-binding sites in heterologously expressed human 13 GABA(A)Rs. Propofol, AziPm, etomidate, and R-mTFD-MPAB each inhibited [H-3]AziPm photoincorporation into GABA(A)R subunits maximally by approximate to 50%. When the amino acids photolabeled by [H-3]AziPm were identified by protein microsequencing, we found propofol-inhibitable photolabeling of amino acids in the 3-1 subunit interface (3Met-286 in 3M3 and 1Met-236 in 1M1), previously photolabeled by [H-3]azietomidate, and 1Ile-239, located one helical turn below 1Met-236. There was also propofol-inhibitable [H-3]AziPm photolabeling of 3Met-227 in M1, the amino acid in the 1-3 subunit interface photolabeled by R-[H-3]mTFD-MPAB. The propofol-inhibitable [H-3]AziPm photolabeling in the GABA(A)R 3 subunit in conjunction with the concentration dependence of inhibition of that photolabeling by etomidate or R-mTFD-MPAB also establish that each anesthetic binds to the homologous site at the 3-3 subunit interface. These results establish that AziPm as well as propofol bind to the homologous intersubunit sites in the GABA(A)R transmembrane domain that binds etomidate or R-mTFD-MPAB with high affinity.