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, 122 (4), 776-86

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

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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

Feng Tao et al. Anesthesiology.

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.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Halothane and isoflurane dose-dependently disrupt PDZ domain-mediated interactions between PSD-95 and potassium channel Kv1.4 in a yeast two-hybrid system
(A and B) The effect of halothane on the growth of yeast cells harboring pGADT7-PSD-95 PDZ2 and pGBKT7-Kv1.4 CT100 (n = 6). (A) Halothane dose-dependently inhibited the yeast growth. (B) Yeast growth is shown as a percent of control, relative to halothane concentration. *p < 0.05 and **p < 0.01 vs. 0.00 mM halothane. (C–E) The effect of isoflurane on the growth of yeast cells harboring pGADT7-PSD-95 PDZ2 and pGBKT7-Kv1.4 CT100 (n = 3). (C) Isoflurane dose-dependently inhibited the yeast growth. (D) Yeast growth is shown as a percent of control, relative to isoflurane concentration. *p < 0.05 vs. 0.00 mM isoflurane. (E) Binned scatter plots of (C). CT100, C-terminal 100 amino acids; PDZ, postsynaptic density protein-95, Drosophila disc large tumor suppressor, and zonula occludens-1; PSD-95, postsynaptic density protein-95.
Fig. 2
Fig. 2. Halothane and isoflurane dose-dependently disrupt the association between PSD-95 and potassium channel Kv1.4 in the forebrain
(A) The GST pull-down assay showed that halothane dose-dependently inhibits the interaction between PSD-95 PDZ2 and Kv1.4 C-terminus. The bottom panels indicate equal amounts of loading of GST-fusion proteins. (B) The co-immunoprecipitation assay showed that isoflurane dose-dependently disrupts the association between PSD-95/SAP90 and Kv1.4 in vivo. The specificity of the anti-Kv1.4 antibody was verified by preincubation with Kv1.4 fusion peptide (N). As a positive control (input), 40 μg of the solubilized membrane proteins were loaded onto the gel. IP, immunoprecipitation; IB, immunoblotting; GST, glutathione S-transferase; PSD-95, postsynaptic density protein-95.
Fig. 3
Fig. 3. Halothane and isoflurane dose-dependently disrupt PDZ domain-mediated interactions between AMPA receptor subunit GluR2 and GRIP or PICK1 in a yeast two-hybrid system
(A–C) The effect of halothane on the growth of yeast cells harboring pGADT7-GluR2 CT and pGBKT7-GRIP PDZ4,5 (n = 3). (A) Halothane dose-dependently inhibited the yeast growth. (B) Yeast growth is shown as a percent of control, relative to halothane concentration. *p < 0.05 vs. 0.00 mM halothane. (C) Binned scatter plots of (A). (D–F) The effect of isoflurane on the growth of yeast cells harboring pGADT7-GluR2 CT and pGBKT7-PICK1 PDZ (n = 3). (D) Isoflurane dose-dependently inhibited the yeast growth. (E) Yeast growth is shown as a percent of control, relative to isoflurane concentration. *p < 0.05 vs. 0.00 mM isoflurane. (F) Binned scatter plots of (D). CT, C-terminus; GRIP, glutamate receptor interacting protein; PICK1, protein interacting with c kinase 1.
Fig. 4
Fig. 4. Isoflurane dose-dependently disrupts PDZ domain-mediated interactions between PSD-95 and NR2B in a yeast two-hybrid system
(A) The effect of isoflurane on the growth of yeast cells harboring pGADT7-PSD-95 PDZ2 and pGBKT7-NR2B C-terminus. Note that isoflurane dose-dependently inhibited the yeast growth. (B) Yeast growth is shown as a percent of control, relative to isoflurane concentration (n = 6). *P < 0.05 and **P < 0.01 vs. 0.00 mM isoflurane. PSD-95, postsynaptic density protein-95.
Fig. 5
Fig. 5. Isoflurane dose-dependently disrupts the association between PSD-95 and NR2B in real time
Surface Plasmon resonance analysis revealed that isoflurane dose-dependently reduces the real-time binding of PSD-95 PDZ2 to NR2B C-terminal peptide, as illustrated by superimposed sensorgrams. RU, resonance units.
Fig. 6
Fig. 6. Halothane has no effect on PDZ domain-mediated protein-protein interactions between GABAB receptor subunit 2 and PAPIN or Mupp1 in a yeast two-hybrid system
(A) No concentration of halothane tested inhibited the growth of yeast cells harboring pGADT7-GABABR2 and pGBKT7-PAPIN PDZ1 or pGBKT7-Mupp1 PDZ13. Consistent with our previous study, halothane dose-dependently inhibited the growth of yeast cells harboring pGADT7-PSD-95 PDZ2 and pGBKT7-NR2B C-terminus, which were used as a positive control. (B) Yeast growth is shown as a percent of control, relative to halothane concentration (n = 6). *p < 0.05 and **p < 0.01 vs. 0.00 mM halothane. PSD-95, postsynaptic density protein-95; GABABR2, GABAB receptor subunit 2.
Fig. 7
Fig. 7. Isoflurane has no effect on PDZ domain-mediated interactions between GABAB receptor subunit 2 and PAPIN or Mupp1 in a yeast two-hybrid system
(A) No concentration of isoflurane tested inhibited the growth of yeast cells harboring pGADT7-GABABR2 and pGBKT7-PAPIN PDZ1 or pGBKT7-Mupp1 PDZ13. Consistent with our previous study, isoflurane dose-dependently inhibited the growth of yeast cells harboring pGADT7-PSD-95 PDZ2 and pGBKT7-NR2B C-terminus, which were used as a positive control. (B) Yeast growth is shown as a percent of control, relative to isoflurane concentration (n = 6). *p < 0.05 and **p < 0.01 vs. 0.00 mM isoflurane. PSD-95, postsynaptic density protein-95; GABABR2, GABAB receptor subunit 2.
Fig. 8
Fig. 8. Isoflurane concentration-dependent changes in chemical shift of the PDZ domains of PSD-95
(A) A representative 15N-1H HSQC spectrum of the 15N-labeled PDZ1–3 of rat source PSD-95 (residues 61–393) showing 230 assigned residues. (B) Representative 1H chemical shift changes of residues in the PDZ1–3 as a function of isoflurane concentrations. (C) Structures of PDZ1–3 mapped with the combined chemical shifts ( Δppm=(ΔδHN)2+(0.2×ΔδN)2) induced by isoflurane. Residues experienced chemical shift changes are highlighted in red (Δδ≧ 0.03 ppm), green (Δδ ~ 0.02–0.03ppm), and orange (Δδ ~ 0.01–0.02 ppm). Isoflurane concentration was calibrated with the external reference of trifluoroacetic acid. PDZ, postsynaptic density protein-95, Drosophila disc large tumor suppressor, and zonula occludens-1.
Fig. 9
Fig. 9. Changes in PDZ1–3 of PSD-95 produced by binding of peptides NR2A-c20 or NR2B-c20 in the absence or presence of isoflurane
(A and B) The structure of PDZ1–3 of PSD-95 showing residues with diminished (left) and enhanced (right) NMR peak intensities after binding to NR2A-c20 (A) or NR2B-c20 (B). Color codes: blue – changed only by NR2A-c20 binding; red- changed only by NR2B-c20 binding; green-changed in both cases of binding. The same color codes are applied to all figures here. (C) The combined 1H and 15N chemical shift changes in PDZ1–3 of PSD-95 upon NR2A-c20 (blue) or NR2B-c20 (red) binding. (D) The combined 1H and 15N chemical shift changes induced by isoflurane in PDZ1–3 of PSD-95 bound with NR2A-c20 (red) or NR2B-c20 (green). (E–G) The structure of PDZ1–3 of PSD-95 showing isoflurane-disturbed residues (Δδ≧ 0.04 ppm) upon binding of NR2A-c20 (E), NR2B-c20 (F) or no peptide present (G). Concentrations used are 100 μM for PDZ1–3 of PSD-95, 310 μM for peptides, and 3 mM for isoflurane. PDZ, postsynaptic density protein-95, Drosophila disc large tumor suppressor, and zonula occludens-1.

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