Mapping the sevoflurane-binding sites of calmodulin

Abstract

General anesthetics, with sevoflurane (SF) being the first choice inhalational anesthetic agent, provide reversible, broad depressor effects on the nervous system yet have a narrow margin of safety. As characterization of low-affinity binding interactions of volatile substances is exceptionally challenging with the existing methods, none of the numerous cellular targets proposed as chief protagonists in anesthesia could yet be confirmed. The recognition that most critical functions modulated by volatile anesthetics are under the control of intracellular Ca(2+) concentration, which in turn is primarily regulated by calmodulin (CaM), motivated us for characterization of the SF-CaM interaction. Solution NMR (Nuclear Magnetic Resonance) spectroscopy was used to identify SF-binding sites using chemical shift displacement, NOESY and heteronuclear Overhauser enhancement spectroscopy (HOESY) experiments. Binding affinities were measured using ITC (isothermal titration calorimetry). SF binds to both lobes of (Ca(2+))4-CaM with low mmol/L affinity whereas no interaction was observed in the absence of Ca(2+). SF does not affect the calcium binding of CaM. The structurally closely related SF and isoflurane are shown to bind to the same clefts. The SF-binding clefts overlap with the binding sites of physiologically relevant ion channels and bioactive small molecules, but the binding affinity suggests it could only interfere with very weak CaM targets.

Keywords: Anesthesia; ITC; NMR; anesthetic binding; calmodulin; sevoflurane.

Figure 1

Representative sections of the ¹H,¹³C-HSQC spectra of titrations of CaM with SF. Sevoflurane (SF, A) titrations to methionine methyl region of apo-CaM (B) and (Ca²⁺)₄-CaM (C) in blue, overlaid with spectra taken after successive additions of 2.4 (magenta), 5.0 (purple), and 10 (red) mmol/L SF.

Figure 2

Structural identification of the sevoflurane (SF)-binding sites to (Ca²⁺)₄-CaM. Surface representation of (Ca²⁺)₄-CaM (pdb id 1X02) (Kainosho et al. 2006) color coded accordingly: amide atoms with Δδ(¹H,¹⁵N)>50 Hz in blue, methyl atoms with Δδ(¹H,¹³C)>50 Hz in magenta, Met Hε protons with NOEs to SF in red and Ca²⁺ ions in black. SF, docked using Glide (Schrödinger, Inc.), is shown in CPK representation. The N-terminal binding site for SF to (Ca²⁺)₄-CaM is shown in (A); a view of the C-terminal binding site is obtained by turning the structure 180 degrees (B). A close-up of the methyl groups of each binding interface with assignments is shown, to the right.

Figure 3

Correlation of the residue-specific chemical shift changes of (Ca²⁺)₄-CaM upon titration with isoflurane and sevoflurane. Residue-specific Δδ (¹H,¹⁵N), in Hz, for 0.2 mmol/L U-[¹³C,¹⁵N]-(Ca²⁺)₄-CaM with 0/10 mmol/L sevoflurane versus Δδ (¹H,¹⁵N), in Hz, for 0.1 mmol/L U-[¹⁵N]-(Ca²⁺)₄-CaM with 0/13 mmol/L isoflurane, is shown as measured for 88 backbone amides.

Figure 4

Determination of the sevoflurane (SF) affinity for the individual (Ca²⁺)₂-loaded CaM lobes by isothermal titration calorimetry (ITC). Representative ITC experiments showing binding of antagonist W-7 with the (Ca²⁺)₂-loaded CaM N-lobe (A) and C-lobe (B) in the presence of 20 mmol/L SF. The upper panels represent ITC raw data with each peak corresponding to an injection event. The lower panels show integrated values for W-7 binding to (Ca²⁺)₂-CaM lobes in the presence (black squares) and absence (open red circles) of 20 mmol/L SF. Integrated values are fit to a two-site binding model, with the first high-affinity site used to determine the affinity of SF. The second binding-site is too weak to quantify accurately. The W-7 binding affinities of (Ca2+)₄-CaM were observed to alter from K_d 5.5 to 18.0 μmol/L and from K_d 15.6 to 32.6 μmol/L for the C and N-lobes, respectively, upon changing the SF concentration from 0 to 20 mmol/L. The aqueous SF concentration (20 mmol/L) of the solutions utilized in the ITC experiments was confirmed by ¹H NMR (Fig. S6). SF binding affinities are given in the text.

Figure 5

The sevoflurane (SF)-dependent Ca²⁺ affinity for the individual lobes assessed by isothermal titration calorimetry (ITC). ITC experiments were utilized to measure the affinity of CaM lobes for Ca²⁺ in the presence and absence of SF. (A) Upper panel: Typical ITC curve for CaM–Ca²⁺ interaction. Lower panel: Overlay of integrated ITC data for the N-lobe. Titrations consisted of 40 injections of 1 μL CaCl₂ at 1.6 mmol/L into the cell containing the N-lobe at a 20-fold lower concentration (0.5 mmol/L), and with SF concentrations at 0 (gray), 0.4 (green), or 20 mmol/L SF (red) The measurement performed with 0.4 mmol/L SF is pharmacologically relevant as it corresponds to the clinical blood concentration of SF at anesthesia (Franks and Lieb ; Streiff et al. 2004). (B) Bar graphs indicating the average ΔG and K_d for the N-lobe binding sites. The error bars indicate standard errors based on three independent measurements for each titration. Two-tailed paired t-test: *P = 0.047; **P = 0.0075.

Figure 6

Superimposed structures of (Ca²⁺)₄-CaM binding sevoflurane (SF), W-7, and ion channel domains. Surface representation of (Ca²⁺)₄-CaM (pdb id 1X02) (Kainosho et al. 2006) N-terminal domain (A, residues 13–80 for clarity) and C-terminal domain (B, residues 81–148). For color coding scheme, see Figure 2. Ligands are shown in stick representation with SF (docked using Glide, Schrödinger, Inc.) in green, W-7 in yellow, skeletal muscle isoform ryanodine receptor (RYR1) peptide representing (residues Arg3629–Met3638 and Ala3618–Lys3626 interacting with the N- and C-terminal domain, respectively) in red and human NaV1.5 DIII-IV linker, residues Asn1489–Lys1500, in blue.