Multiple propofol-binding sites in a ?-aminobutyric acid type A receptor (GABAAR) identified using a photoreactive propofol analog.

ABSTRACT: Propofol acts as a positive allosteric modulator of ?-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. [(3)H]2-(3-Methyl-3H-diaziren-3-yl)ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate (azietomidate) and R-[(3)H]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 GABAAR transmembrane domain. Here, we use a photoreactive analog of propofol (2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol ([(3)H]AziPm)) to identify propofol-binding sites in heterologously expressed human ?1?3 GABAARs. Propofol, AziPm, etomidate, and R-mTFD-MPAB each inhibited [(3)H]AziPm photoincorporation into GABAAR subunits maximally by ? 50%. When the amino acids photolabeled by [(3)H]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 [(3)H]azietomidate, and ?1Ile-239, located one helical turn below ?1Met-236. There was also propofol-inhibitable [(3)H]AziPm photolabeling of ?3Met-227 in ?M1, the amino acid in the ?1-?3 subunit interface photolabeled by R-[(3)H]mTFD-MPAB. The propofol-inhibitable [(3)H]AziPm photolabeling in the GABAAR ?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 GABAAR transmembrane domain that binds etomidate or R-mTFD-MPAB with high affinity.

SUBMITTER: Jayakar SS 

PROVIDER: S-EPMC4183786 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature