Isoflurane binds and stabilizes a closed conformation of the leukocyte function-associated antigen-1

We previously demonstrated that isoflurane targets lymphocyte function-associated antigen-1 (LFA-1), a critical adhesion molecule for leukocyte arrest. However, it remains to be determined how isoflurane interacts with the full ectodomain LFA-1 and modulates its conformation and function. Isoflurane binding sites on the full ectodomain LFA-1 were probed by photolabeling using photoactivatable isoflurane (azi-isoflurane). The adducted residues were determined by liquid chromatography/mass spectrometry analysis. Separately, docking simulations were performed to predict binding sites. Point mutations were introduced around isoflurane binding sites. The significance of isoflurane's effect was assessed in both intracellular adhesion molecule-1 (ICAM-1) binding assays and epitope mapping of activation-sensitive antibodies using flow cytometry. Two isoflurane binding sites were identified using photolabeling and were further validated by the docking simulation: one at the hydrophobic pocket in the ICAM-1 binding domain (the alpha I domain); the other at the beta I domain. Mutagenesis of the alpha'1 helix showed that isoflurane binding sites at the beta I domain were significantly important in modulating LFA-1 function and conformation. Epitope mapping using activation-sensitive antibodies suggested that isoflurane stabilized LFA-1 in the closed conformation. This study suggested that isoflurane binds to both the alpha I and beta I domains allosteric to the ICAM-1 binding site, and that isoflurane binding stabilizes LFA-1 in the closed conformation.-Yuki, K., Bu, W., Xi, J., Sen, M., Shimaoka, M., Eckenhof, R. G. Isoflurane binds and stabilizes a closed conformation of the leukocyte function-associated antigen-1. FASEB J. 26, 4408-4417 (2012). www.fasebj.org