Deuterium NMR and high-pressure FT-IR studies of membranes: Anesthetic-lipid interactions and molecular dynamics in lipid bilayers.

Abstract

The interactions of the local anesthetic tetracaine with multilamellar dispersions of dimyristoylphosphatidylcholine (DMPC) containing cholesterol have been investigated by deuterium nuclear magnetic resonance ($\sp2$H NMR). $\sp2$H NMR spectra of tetracaine indicate that the location of the anesthetic in the cholesterol-containing DMPC bilayers differs from that in pure phosphatidylcholine bilayers, the anesthetic being located closer to the lipid-water interface in the former system. Moreover, the incorporation of the anesthetic into DMPC bilayers with or without cholesterol results in a reduction of the lipid order parameters both in the plateau and in the tail regions of the acyl chains, but does not significantly affect the cholesterol ordering. The interactions of tetracaine with the glycolipid 1,2-di-O-tetradecyl-3-O-($\beta$-D-glucopyranosyl)-sn-glycerol ($\beta$-DTGL) and with $\beta$-DTGL (20 mole%) in DMPC have also been investigated by $\sp2$H NMR. The stability of the lamellar structure of the pure glycolipid system is very sensitive to the presence of anesthetic while the interaction of tetracaine with the mixed glycolipid-phospholipid system does not trigger the formation of non-lamellar phases but leads to a slight reduction in molecular ordering. The location of tetracaine in different lipid bilayers and in nerves has been studied by high-pressure Fourier transform infrared (FT-IR) spectroscopy. The results reveal a correlation between the location of the anesthetic in model membranes and that in nerves. They also indicate that tetracaine is expelled by pressure from both model and nerve membranes, and that for model membrane systems, low pH or cholesterol will assist pressure in squeezing the anesthetic out of the bilayer. On the other hand, high-pressure FT-IR has also been used to study the effects of tetracaine on the structural and dynamic properties of lipids in model membrane systems. A combination of $\sp2$H spin-lattice relaxation and lineshape analysis has been used to demonstrate that a simple model involving two motions is sufficient to describe the spectral and relaxation features of the glycerol-labelled glycolipid $\beta$-DTGL. The lineshape and relaxation features of this lipid in the gel phase are best simulated using the three-site jump model with relative site populations of 0.46, 0.34 and 0.20, and a correlation time of 6.7 $\times$ 10$\sp{-10}$s. A second motion, namely rotation about the long axis of the molecule as a whole, is needed to account for the observed variation in the quadrupolar echo amplitude and the spectral lineshape over the temperature range of 25 to 60$\sp\circ$C. Similar results have been obtained for several phospholipid and glycolipid bilayers, which suggest that the glycerol backbone dynamics in all these systems can be described in terms of common fast internal motions and a slower whole molecule axial motion. Two-dimensional solid-state deuteron NMR spectroscopy has been used to confirm the presence of a slow whole molecule motion in the gel phase of the glycolipid $\beta$-DTGL at 35$\sp\circ$C, with an associated correlation time of the order of milliseconds. Comparison of the experimental and simulated two-dimensional ridge patterns suggest that a large angle jump about the long molecular axis can best account for the 2D exchange spectra of $\beta$-DTGL in the gel phase in comparison to small step Brownian diffusion. On the other hand, it is demonstrated that lateral diffusion over curved membrane surface of dipalmitoylphosphatidylcholine bilayers in the liquid-crystalline phase can be detected by 2D deuteron NMR, with an associated correlation time of the order of $\approx$100 ms.