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 ␣1␤3 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.

The γ-aminobutyric acid type-A receptor (GABAAR) is a target for general anesthetics of diverse chemical structures, which act as positive allosteric modulators at clinical doses. Previously, in a heterogeneous mixture of GABAARs purified from bovine brain, [3H]azietomidate photolabeling of αMet-236 and βMet-286 in the αM1 and βM3 transmembrane helices identified an etomidate binding site in the GABAAR transmembrane domain at the interface between the β and α subunits. To further define GABAAR etomidate binding sites, we now use [3H]TDBzl-etomidate, an aryl diazirine with broader amino acid side-chain reactivity than azietomidate, to photolabel purified human FLAG-α1β3 GABAARs and obtain a more extensive identification of photolabeled GABAAR amino acids. [3H]TDBzl-etomidate photolabeled in an etomidate-inhibitable manner β3Val-290, in the β3M3 transmembrane helix, as well as α1Met-236 in α1M1, a residue photolabeled by [3H]azietomidate, while no photolabeling was detected of amino acids in the αM2 or βM2 helices that also border the etomidate binding site. The location of these photolabeled amino acids in GABAAR homology models derived from the recently solved structures of prokaryote (GLIC) or invertebrate (GluCl) homologs and the results of computational docking studies predict the orientation of [3H]TDBzl-etomidate bound in that site and the other amino acids contributing to this GABAAR intersubunit etomidate binding site. Etomidate-inhibitable photolabeling of β3Met-227 in βM1 by [3H]TDBzl-etomidate and [3H]azietomidate also provides evidence of an homologous etomidate binding site at the β3-β3 subunit interface in the α1β3 GABAAR.

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Summary Spermadhesins are a novel family of secretory proteins expressed in the male genital tract of pig, horse and bull. They are major products of the seminal plasma and have been found to be peripherally associated to the sperm surface. The structure and function of spermadhesins have been thoroughly investigated in the pig, which exhibits the greatest diversity of members: AWN, AQN‐1, AQN‐2, PSP‐I and PSP‐II and its glycosylated isoforms. They are multifunctional proteins showing a range of ligand‐binding abilities, e.g. carbohydrates, sulfated glycosamino‐glycans, phospholipids and protease inhibitors, suggesting that they may be involved in different steps of fertilization. Isolated porcine spermadhesins bind the zona pellucida glycoproteins in a cation‐dependent manner with a Kd in a low micromolar range, and AWN, AQN‐1 and AQN‐3 display similar binding affinity for glyoproteins containing Galβ(1–3)‐GalNAc and Galβ(1–4)‐GlcNAc sequences in O‐linked and N‐linked oligosaccharides, respectively. During sperm passage through the epididymis AQN‐3 and AWN have been shown to bind tightly to the sperm surface by interaction with the phospholipids of the membrane bilayer. At ejaculation the spermadhesins form a protective coat around the sensitive acrosomal region of the sperm head, thus possibly preventing premature acrosome reaction. During in vitro capacitation most of these aggregated sperm adhesins are lost, with the exception of phospholipid‐bound spermadhesins. AWN and AQN‐3 may now serve as a primary receptor for the oocyte zona pellucida, thus contributing to initial binding and recognition between sperm and egg. The amino acid sequence of spermadhesins does not show any discernible similarity with known carbohydrate recognition domains (CRD). However, they belong to the superfamily of proteins with a CUB domain with a predicted all‐β structure. The crystal structure of the heterodimeric complex of the spermadhesins PSP‐I/PSP‐II has been solved, showing that the overall structure of both spermadhesins consists of a β‐sandwich with five (parallell and antiparallel) β‐strands. It is the first three‐dimensional structure of a zona pellucida‐binding protein and reveals the architecture of the CUB domain. The spermadhesins represent a novel class of lectins that may be involved in sequential steps of fertilization, at least in the pig.