Massachusetts General Hospital
Department of Anesthesia
Boston, MA 02114
tel: (617) 726-8985 fax: (617) 726-5845
email: k_miller@helix.mgh.harvard.edu
The interest of my laboratory is the role of the lipid environment on the function of membrane proteins such as ion channels. Such studies are hampered because X-ray diffraction and NMR can only be applied with great difficulty. We approach this problem using Electron Spin Resonance and Fourier Transform Infra-red (FTIR) spectroscopy to obtain information about structure, and rapid kinetics techniques to obtain information about function. The role of lipid is dissected by purifying proteins and incorporating them into bilayers of defined composition. Currently, the main subject of our studies is the nicotinic acetylcholine receptor, an intrinsic membrane protein which may be obtained at high specific activity and in high yield (tens of milligram quantities) from the electric tissue of certain fish. This receptor is part of a "super-family" that includes the GABA, glycine and 5HT3 receptors, so that work on it is of wide interest. The function of the receptor is assessed using stopped-flow fluorimetry with either a fluorescent acetylcholine derivative or fluorescent allosteric effectors. We have recently found that the receptor can only be activated in bilayers that contain cholesterol and negatively charged lipids. The reasons for this behavior are sought using structural techniques. For example, lipid-protein interactions are studied with spin-labeled probes attached to lipids and to the receptor; such studies show that cholesterol and negatively charged lipids have a preference for the lipid-protein interface. Structural changes accompanying exposure to acetylcholine are explored in a collaboration using FTIR difference spectroscopy. A related focus of the laboratory is the mechanism of action of membrane acting drugs such as general anesthetics and ethanol which may act by perturbing lipid-protein interactions or interacting with hydrophobic regions of the ion channel. We find that their actions are dependent on the lipid bilayer surrounding the receptor.
Selected Publications:
Raines, D.E., Miller, K.W. (1993). The Role of Charge in Lipid Selectivity for the Nicotinic Acetylcholine Receptor. Biophysics Journal 64: 632-641.
Abadji, V.C., Raines, D.E., Watts, A., Miller, K.W. (1993). The Effect of General Anesthetics on the Dynamics of Phosphatidylcholine-Acetylcholine Receptor Interactions in Reconstituted Vesicles. Biochem. Biophys, Acta 1147: 143-153.
Baenziger, J.E., Miller, K.W., Rothschild, K.J. (1993). Fourier Transform Infrared Difference Spectroscopy of the Nicotinic Acetylcholine Receptor: Evidence for Special Protein Structural Changes Upon Desensitization. Biochemistry 32: 5448-5454.