Three classes of propofol binding sites on GABAA receptors

Propofol is a widely used anesthetic and sedative that acts as a positive allosteric modulator of gamma-aminobutyric acid type A (GABAA) A ) receptors. Several potential propofol binding sites that may mediate this effect have been identified fi ed using propofol-analogue photoaffinity fi nity labeling. Ortho-propofol diazirine (o-PD) labels 13-H267, a pore-lining residue, whereas AziPm labels residues 13-M286, 13-M227, and a-I239 in the two membrane-facing interfaces [13(+)/a(-) 13 (+)/ a ( - ) and a (+)/ 13 ( - )] between a and 13 subunits. This study used photoaffinity fi nity labeling of a1133 1 13 3 GABAA A receptors to reconcile the apparently conflicting fl icting results obtained with AziPm and o-PD labeling, focusing on whether 13 3-H267 identifies fi es specific fi c propofol binding site(s). The results show that propofol, but not AziPm protects 13 3-H267 from labeling by o-PD, whereas both propofol and o-PD protect against AziPm labeling of 13 3-M286, 13 3-M227, and a 1 I239. These data indicate that there are three distinct classes of propofol binding sites, with AziPm binding to two of the classes and o-PD to all three. Analysis of binding stoichiometry using native mass spectrometry in 133 3 homomeric receptors, demonstrated a minimum of fi ve AziPm labeled residues and three o-PD labeled residues per pentamer, suggesting that there are two distinct propofol binding sites per 13-subunit. The native mass spectrometry data, coupled with photolabeling performed in the presence of zinc, indicate that the binding site(s) identified fi ed by o-PD are adjacent to, but not within the channel pore, since the pore at the 170 0 H267 residue can accommodate only one propofol molecule. These data validate the existence of three classes of specific fi c propofol binding sites on a1133 1 13 3 GABAA A receptors.