Inhalational Anesthetics Disrupt Postsynaptic Density Protein-95, Drosophila Disc Large Tumor Suppressor, and Zonula Occludens-1 Domain Protein Interactions Critical to Action of Several Excitatory Receptor Channels Related to Anesthesia

Abstract

Abstract Background: The authors have shown previously that inhaled anesthetics disrupt the interaction between the second postsynaptic density protein-95, Drosophila disc large tumor suppressor, and zonula occludens-1 (PDZ) domain of postsynaptic density protein-95 (PSD-95) and the C-terminus of N-methyl-d-aspartate receptor subunits NR2A and NR2B. The study data indicate that PDZ domains may serve as a molecular target for inhaled anesthetics. However, the underlying molecular mechanisms remain to be illustrated. Methods: Glutathione S-transferase pull-down assay, coimmunoprecipitation, and yeast two-hybrid analysis were used to assess PDZ domain–mediated protein–protein interactions in different conditions. Nuclear magnetic resonance spectroscopy was used to investigate isoflurane-induced chemical shift changes in the PDZ1–3 domains of PSD-95. A surface plasmon resonance–based BIAcore (Sweden) assay was used to examine the ability of isoflurane to inhibit the PDZ domain–mediated protein–protein interactions in real time. Results: Halothane and isoflurane dose-dependently inhibited PDZ domain–mediated interactions between PSD-95 and Shaker-type potassium channel Kv1.4 and between α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit GluA2 and its interacting proteins—glutamate receptor–interacting protein or protein interacting with c kinase 1. However, halothane and isoflurane had no effect on PDZ domain–mediated interactions between γ-aminobutyric acid type B receptor and its interacting proteins. The inhaled anesthetic isoflurane mostly affected the residues close to or in the peptide-binding groove of PSD-95 PDZ1 and PDZ2 (especially PDZ2), while barely affecting the peptide-binding groove of PSD-95 PDZ3. Conclusion: These results suggest that inhaled anesthetics interfere with PDZ domain–mediated protein–protein interactions at several receptors important to neuronal excitation, anesthesia, and pain processing.