摘要：

Propofol acts as a positive allosteric modulator of γ-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. [ 3 H]2-(3-Methyl-3 H -diaziren-3-yl)ethyl 1-(phenylethyl)-1 H -imidazole-5-carboxylate (azietomidate) and R -[ 3 H]5-allyl-1-methyl-5-( m -trifluoromethyl-diazirynylphenyl)barbituric acid ( m TFD-MPAB), photoreactive analogs of 2-ethyl 1-(phenylethyl)-1 H -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)-3 H -diazirin-3-yl]phenol ([ 3 H]AziP m )) to identify propofol-binding sites in heterologously expressed human α1β3 GABA A Rs. Propofol , AziP m , etomidate , and R-m TFD-MPAB each inhibited [ 3 H]AziP m photoincorporation into GABA A R subunits maximally by 50%. When the amino acids photolabeled by [ 3 H]AziP m 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 [ 3 H]azietomidate, and α1Ile-239, located one helical turn below α1Met-236. There was also propofol-inhibitable [ 3 H]AziP m photolabeling of β3Met-227 in βM1, the amino acid in the α1-β3 subunit interface photolabeled by R -[ 3 H] m TFD-MPAB. The propofol-inhibitable [ 3 H]AziP m photolabeling in the GABA A R β3 subunit in conjunction with the concentration dependence of inhibition of that photolabeling by etomidate or R-m TFD-MPAB also establish that each anesthetic binds to the homologous site at the β3-β3 subunit interface. These results establish that AziP m as well as propofol bind to the homologous intersubunit sites in the GABA A R transmembrane domain that binds etomidate or R-m TFD-MPAB with high affinity.

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