The volatile anesthetic isoflurane perturbs conformational activation of integrin LFA-1 by binding to the allosteric regulatory cavity

Abstract

The molecular and structural basis of anesthetic interactions with conformations and functionalities of cell surface receptors remains to be elucidated. We have demonstrated that the widely used volatile anesthetic isoflurane blocks the activation-dependent conformational conversion of integrin lymphocyte function associated antigen-1 (LFA-1), the major leukocyte cell adhesion molecule, to a high-affinity configuration. Perturbation of LFA-1 activation by isoflurane at clinically relevant concentrations leads to the inhibition of T-cell interactions with target cells as well as ligand-triggered intracellular signaling. Nuclear magnetic resonance spectroscopy reveals that isoflurane binds within a cavity in the LFA-1 ligand-binding domain, which is a previously identified drug-binding site for allosteric small-molecule antagonists that stabilize LFA-1 in a low-affinity conformation. These results provide a potential mechanism for the immunomodulatory properties of isoflurane.

Figure 1.

A model for LFA-1 activation. A) Graphic drawings showing the conversion of the LFA-1 I domain from the low-affinity (left) to the high-affinity (middle) conformation, which is triggered by a downward shift of the C-terminal helix (shown as an arrow). A small-molecule allosteric LFA-1 antagonist such as lovastatin (gray triangle) binds underneath the C-terminal helix and perturbs the conversion to the high-affinity conformation (right). The body of the I domain is shown as a box, the ligand binding site at the top of the domain as a circle, and the C-terminal helix as a zigzag line. Inactive and active states are shown in blue and red, respectively. B) Global conformational changes of LFA-1 from the bent form (left, in the absence of activation) to the extended form (right, in the presence of activation). The extracellular domains are shown as graphic drawings: low-affinity I domain (blue), high-affinity I domain (red), α-subunit not containing the I domain (white), and β-subunit (gray). The plasma membrane is shown by two parallel dashed lines. Transmembrane and cytoplasmic domains for α and β subunits are shown in gray and back, respectively. The position of the KIM127 activation-dependent epitope is shown in yellow. Note that the KIM127 epitope was accessible only in the extended LFA-1.

Figure 2.

Isoflurane at clinically relevant concentrations inhibits LFA-1 function. A–C) Isoflurane blocked ICAM-1 binding to LFA-1 (A, B) without altering its cell surface expression probed by mAb TS1/22 (C) and TS1/18 (data not shown), as determined by flow cytometry on PBMCs, Jurkat T-cell line, and K562 transfectant expressing WT LFA-1 (K-LFA-1). D) Isoflurane (1.5 mM) suppressed LFA-1-ICAM-1-dependent cell-cell conjugate formation of Jurkat and U937 cells. E) Isoflurane (1.5 mM) suppressed Mn²⁺-induced transition of LFA-1 from the bent to the extended conformation, as shown by the inhibition of KIM127 epitope exposure. MFI, mean fluorescence intensity. F) Isoflurane blocked phosphorylation of ERK in Mn²⁺-stumulated Jurkat cells induced by ICAM-1 but not by mAb TS1/22. Cells were treated for 30 min with ICAM-1 (5 μg/ml) or TS 1/22 (5 μg/ml) in the absence or presence of isoflurane. Cell lysate was subjected to Western blot analysis probed with anti-p44/42 (ERK) or anti-phospho p44/42 (p-ERK) antibodies. A representative result is shown. Data are expressed as means ± se of 3 independent experiments. *P < 0.05 vs. mock-treated samples.

Figure 3.

Isoflurane inhibits LFA-1 allosterically. A) Binding of ICAM-1 to WT and locked high-affinity LFA-1 K287C/K294C (KK/CC LFA-1) on K562 cells was examined using flow cytometry in the presence or absence of different concentrations of isoflurane. Isoflurane failed to block locked KK/CC LFA-1; however, on DTT reduction, isoflurane blocked Mn²⁺-stimulated KK/CC LFA-1 as well as Mn²⁺-stimulated WT LFA-1. B) Isoflurane inhibited ICAM-1 binding to LFA-1 in a cell-free system. Binding of ICAM-1 to the extracellular part of WT LFA-1 or KK/CC LFA-1 immobilized on plates was studied using a colorimetric ELISA-type assay while exposed to different concentrations of isoflurane in a vaporous closed chamber. Bound ICAM-1 was detected colorimetrically at an optical density (OD) of 405 nm using peroxidase-labeled goat anti-human Fc and substrate. Data represent means ± se of 3 independent experiments and are expressed as percentage of Mock-treated samples. *P < 0.05 vs. other treatments at the same isoflurane concentration.

Figure 4.

NMR spectroscopy to study isoflurane-binding sites in the LFA-1 I domain. A) Overlay experiments of ¹H,¹⁵N-HSQC acquired without (blue) and with (red) 12 mM isoflurane. Inset: isoflurane titration series of T291 (top right) and S245 (bottom left). Colors correspond to: 0 mM (blue), 3 mM (purple), 6 mM (orange), 9 mM (yellow), and 12 mM (red) isoflurane. B) Scaled chemical-shift perturbation of 12 mM isoflurane mapped onto the LFA-1 I domain protein sequence and secondary structure. Chemical-shift perturbation calculated as [0.2(δ_N^iso − δ_N⁰)²+(δ_H^iso − δ_H⁰)²]^1/2. Inset residues from A are shown in blue (S245) and red (T291), respectively. Secondary structure assignments : β1 (130–137), α1 (144–160), β2 (166–173), β2′ (177–181), α2 (183–188), α3 (192–196), α4 (208–218), β3 (231–238), α5 (249–251), β4 (255–261), α6 (268–277), β5 (286–289), α7 (293–305). For comparison, residues that shifted on addition of the LFA-1 allosteric inhibitor lovastatin are highlighted in yellow . C) Structure of the LFA-1 I domain (residues 127–307, PDB file 1ZOP; ref. 36) showing amide nitrogen residues affected (δ_ppm≥0.05 ppm) by the addition of 12 mM isoflurane. Gray represents residues unperturbed by isoflurane; red represents residues that met or exceeded the threshold for perturbation. Helices and strands are labeled. Residues T291 (red) and S245 (green) are labeled. Yellow spheres represent the Mg²⁺ ion at the ICAM-1 binding site, termed the metal ion-dependent adhesion site (MIDAS). Note that the residues near the MIADS were not affected and that the affected residues clustered near the cavity formed between the α1 and α7 helices and the central β strands. This figure was created using PYMOL.

Figure 5.

Isoflurane inhibits ICAM-1 binding to Mac-1. Binding of ICAM-1-Fc to immobilized extracellular part of WT Mac-1 in the presence or absence of 5% isoflurane was studied by an ELISA-type assay as in Fig. 3B. Bound ICAM-1 was detected colorimetrically at an OD of 405 nm using peroxidase-labeled goat anti-human Fc and substrate. Isoflurane suppressed ICAM-1 binding to Mn²⁺-activated Mac-1 to the basal level (i.e., binding to unactivated Mac-1). ICAM-1 binding was specific to Mac-1, as a background binding of ICAM-1 [i.e., Mac-1 (–)] was negligible. Data are mean and difference from the mean of triplicate samples in two independent experiments.