Multiple Propofol Binding Sites in a {gamma}-Aminobutyric Acid Type A Receptor (GABAAR) Identified Using a Photoreactive Propofol Analog [Molecular Biophysics]

August 1st, 2014 by Jayakar, S. S., Zhou, X., Chiara, D. C., Dostalova, Z., Savechenkov, P. Y., Bruzik, K. S., Dailey, W. P., Miller, K. W., Eckenhoff, R. G., Cohen, J. B.

Propofol acts as a positive allosteric modulator of γ-aminobutyric acid type A receptors (GABAARs), 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 GABAAR modulation. [3H]Azietomidate and [3H]R-mTFD-MPAB, photoreactive analogs of etomidate and mephobarbital, respectively, have identified two homologous but pharmacologically distinct classes of intersubunit binding sites for general anesthetics in the GABAAR transmembrane domain. Here, we use a photoreactive analog of propofol ([3H]AziPm, 2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol) to identify propofol binding sites in heterologously expressed human α1β3 GABAARs. Propofol, AziPm, etomidate, and R-mTFD-MPAB each inhibited [3H]AziPm photoincorporation into GABAAR subunits maximally by ~50%. When the amino acids photolabeled by [3H]AziPm were identified by protein microsequencing, we found propofol-inhibitable photolabeling of amino acids in the β3-α1 subunit interface (β3Met-286 in β3M3 and α1Met-236 in α1M1), previously photolabeled by [3H]azietomidate, and α1Ile-239, located one helical turn below α1Met-236. There was also propofol-inhibitable [3H]AziPm photolabeling of β3Met-227 in βM1, the amino acid in the α1-β3 subunit interface photolabeled by [3H]R-mTFD-MPAB. The propofol-inhibitable 3H]AziPm photolabeling in the GABAAR β3 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 β3-β3 subunit interface. These results establish that AziPm as well as propofol bind to the homologous intersubunit sites in the GABAAR transmembrane domain that bind etomidate or R-mTFD-MPAB with high affinity.
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