Biophys J, February 2000, p. 746-751, Vol. 78, No. 2

NMR Study of Volatile Anesthetic Binding to Nicotinic Acetylcholine Receptors

Yan Xu,^* Tomoyoshi Seto,^* Pei Tang,^* and Leonard Firestone^*

 ^*Department of Anesthesiology and Critical Care Medicine and  Department of Pharmacology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261 USA

New lines of evidence suggest that volatile anesthetics interact specifically with proteins. Direct binding analysis, however, has been largely limited to soluble proteins. In this study, specific interaction was investigated between isoflurane, a clinically important volatile anesthetic, and membrane-bound nicotinic acetylcholine receptors (nAChRs) from Torpedo electroplax, using ¹⁹F nuclear magnetic resonance spectroscopy and gas chromatography. The receptors were reconstituted into 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid vesicles. After correcting for nonspecific partitioning into the lipid, the equilibrium dissociation constant, K_d, of isoflurane binding to nAChR at 15°C was found to be 0.36 ± 0.03 mM. This value is within the clinically relevant concentration range of the agent. Based on the receptor concentrations in the vesicle suspension assayed by the bicinchoninic acid method and the fraction of bound isoflurane, X_b, determined by gas chromatography, an estimate of an average of 9-10 specifically bound isoflurane molecules can be made for each receptor, or two for each subunit. Upon binding, the transverse relaxation time constant (T₂) of ¹⁹F resonance of isoflurane is decreased by nearly three orders of magnitude, indicating a dramatic reduction in the mobility of specifically bound isoflurane. Kinetic analysis reveals that the off rate of binding, k₁, is 1.7 × 10⁴ s¹. The on rate, k₊₁, can thus be calculated to be ~4.8 × 10⁷ M¹ s¹, suggesting a nearly diffusion-limited association. This is in contrast to anesthetic binding to a soluble protein, bovine serum albumin (BSA), where k₊₁ and k₁ are at least an order of magnitude slower. It is concluded that the presence of lipids may be critical for the correct evaluation of binding kinetics between volatile anesthetics and neuronal receptors.

Biophys J, February 2000, p. 746-751, Vol. 78, No. 2
© 2000 by the Biophysical Society   0006-3495/00/02/746/06  $2.00

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