Synaptic PDZ Domain-mediated Protein Interactions Are Disrupted by Inhalational Anesthetics

Abstract

Anesthetics exert multiple effects on the central nervous system through altering synaptic transmission, but the mechanisms for this process are poorly understood. PDZ domain-mediated protein interactions play a central role in organizing signaling complexes around synaptic receptors for efficient signal transduction. We report here that clinically relevant concentrations of inhalational anesthetics dose-dependently and specifically inhibit the PDZ domain-mediated protein interaction between PSD-95 or PSD-93 and the N-methyl-Daspartate receptor or neuronal nitric-oxide synthase. These inhibitory effects are immediate, potent, and reversible and occur at a hydrophobic peptide-binding groove on the surface of the second PDZ domain of PSD-95 in a manner relevant to anesthetic action. These findings reveal the PDZ domain as a new molecular target for inhalational anesthetics.Inhalational anesthetics have been in widespread use for more than 150 years and have been essential in the development of modern surgical procedures, but their molecular mechanisms have remained poorly understood. Early hypotheses based on nonspecific interactions of lipid-soluble anesthetics with the lipid bilayer of neuronal membranes have largely given way to the recent suggestion that anesthetics interact with multiple membrane-associated proteins involved in synaptic transmission (1-5). Inhibitory GABA A and glycine receptors and excitatory N-methyl-D-aspartate (NMDA), 1 nicotinic acetylcholine, and serotonin receptors have been demonstrated as possible physiological targets that underlie general anesthesia (6). Inhalational anesthetics have been shown to both enhance inhibitory receptor-mediated synaptic neurotransmission and depress excitatory receptor-mediated synaptic neurotransmission.Recent studies have revealed a much more complicated picture of excitatory receptor-mediated synaptic transmission than previously anticipated. For efficient synaptic transmission, the downstream effectors are targeted to the receptors by scaffolding proteins via a complex network of protein-protein interactions (7). The PDZ domain is one of the most common protein-protein recognition modules that have been found in diverse scaffolding and signaling proteins (8, 9). The name PDZ derives from the first three proteins (PSD-95/SAP90, Dlg, and ZO-1 (10)) in which these domains were identified. The PDZ domain recognizes specific C-terminal motifs found in target proteins, most often in the cytoplasmic tails of transmembrane receptors and channels (10, 11). The PDZ domain can also recognize structure-related internal motifs to form homo-and heteromeric PDZ-PDZ interactions (12-15). Therefore, PDZ domain-mediated protein-protein interactions provide a framework for the assembly of multiprotein signaling complexes at synapses and neuromuscular junctions. These interactions coordinate and guide the flow of regulatory information and regulate receptor and ion channel activities (16 -19).One of the best understood PDZ domain proteins at synapses is PSD-95, a modula...

Effect of Disrupting N -Methyl-d-aspartate Receptor–Postsynaptic Density Protein-95 Interactions on the Threshold for Halothane Anesthesia in Mice

“…PDZ domainmediated protein interactions play a central role in organizing signaling complexes around synaptic receptors for efficient signal transduction. Our previous studies have demonstrated that halothane dose-dependently and reversibly inhibits PSD-95 PDZ domain-mediated protein interactions and that the halothane binding site on PSD-95 PDZ2 completely overlaps with the binding pocket of PSD-95 for NMDAR NR2 subunits, 15 suggesting a new concept that affecting PDZ domain-mediated protein interactions at synapses in the CNS might be one of molecular mechanisms by which the general anesthetic state is achieved. By knocking down PSD-95 expression in the spinal cord, we have shown that the deficiency of spinal PSD-95 reduced isoflurane MAC in rats.…”

“…14 Our previous studies have shown that clinically relevant concentrations of inhalational anesthetics dosedependently and specifically inhibit the PDZ domainmediated protein interaction between PSD-95 and NMDARs. 15 These inhibitory effects are immediate, potent, and reversible and occur at a hydrophobic peptide-binding groove on the surface of the PSD-95 PDZ2 in a manner relevant to anesthetic action. 15 These findings reveal the PDZ domain as a new molecular target for inhalational anesthetics.…”

“…15 These inhibitory effects are immediate, potent, and reversible and occur at a hydrophobic peptide-binding groove on the surface of the PSD-95 PDZ2 in a manner relevant to anesthetic action. 15 These findings reveal the PDZ domain as a new molecular target for inhalational anesthetics. We have also found that PSD-95 knockdown significantly reduced MAC for isoflurane and attenuated the NMDAinduced increase in isoflurane MAC.…”

“…15 Here, we used subcloning to construct a Tat-PSD-95 PDZ2 plasmid by inserting PSD-95 PDZ2 cDNA into the pTAT-HA expression vector, which contains an amino-terminal, in-frame, 11-amino acid, minimal transduction domain (residues 47-57 of human immunodeficiency virus Tat protein) termed Tat. 17 Two control plasmids were also constructed: mutated Tat-PSD-95 PDZ2, in which three sites critical for interactions between NMDARs and PSD-95 were mutated (K165T, L170R, and H182L), and PSD-95 PDZ2, which contained the same sequences as Tat-PSD-95 PDZ2 but without Tat PTD.…”

Clinically Relevant Concentration Determination of Inhaled Anesthetics (Halothane, Isoflurane, Sevoflurane, and Desflurane) by 19F NMR

“…The effect of anesthetics on the central nervous system is believed to alter synaptic transmission, but the molecular processes are poorly understood [10]. There is evidence that this action may result because of anesthetic-protein interactions [11].…”