BIOPHYSICS SEMINAR Featuring Keith Miller, "Molecular Mechanisms of General Anesthetics; from Lipids to Channels”
The major focus is on molecular mechanisms by which general anesthetics cause anesthesia and side effects . It is challenging to understand how an agent as simple as xenon can cause loss of consciousness. A related project is concerned with mechanisms of fetal alcohol toxicity. Three Current projects focus on general anesthetic– and alcohol–protein interactions. Rotation projects in my laboratory might use techniques such as stopped flow kinetics, site-directed spin labeling, mass spectrometry, time resolved photolabeling or crystallography.
1. General anesthetics interact with the ligand-gated ion channel superfamily of receptors. General anesthetic binding sites on the nicotinic acetylcholine receptor are located using agents that are both general anesthetics and photoaffinity labels (refs 3 & 4). Because general anesthetics tend to act primarily on the open, ion conducting, conformation of these channels and these conformations only exist for milliseconds after activation , we have developed an apparatus that can freeze the channels in the open state (ref 1). They can then be photolabeled and their sites of photoincorporation discovered at leisure.
2. Some, general anesthetics also interact with proteins that indirectly modulate channels. We have photolabeled a site on protein kinase C near one of the diacylglycerol binding sites (ref 2). Currently we are crystallizing subdomains of PKC, and have solved one structure in the presence of general anesthetics. The latter work is done in collaboration with Dr. Stehle.
3. We are examining the hypothesis that the fetal alcohol syndrome may be caused by alcohol binding sites on cell adhesion proteins in the nervous system. Initial photolabel work has determined that there are alcohol–binding sites and the next step is to locate them.
In unrelated work, the parathyroid receptor’s secondary structure and dynamics are being studied by site-directed spin labeling.
Chiara, DC, Kloczewiak, MA, Addona, GH, Yu, JA, Cohen JB, Miller, KW, Site of Resting State Inhibition of the Nicotinic Acetylcholine Receptor by a Hydrophobic Inhibitor. Biochemistry. (2001); 40: 296-304.
Das, J, Addona, GH, Sandberg, WS, Husain, SS, Stehle, T, Miller, KW. Identification of a General Anesthetic Binding Site in the Diacylglycerol-Binding Domain of Protein Kinase C. J. Biol. Chem. (2004); (published online 7/04)
Husain SS, Forman SA, Kloczewiak MA, Addona GH, Olsen RW, Pratt MB, Cohen JB, Miller KW. Synthesis and Properties of 3-(2-Hydroxyethyl)-3-n-pentyldiazirine, a Photoactivable General Anesthetic, Journal of Medicinal Chemistry (1999): 42:3330-3307.
Husain, SS, Zeibel, MR, Ruesch, D, Hong, F, Arevalo, E, Kosterlitz, JA, Olsen RW, Forman, SA, Cohen, JB, Miller, KW. 2-(3-Methyl-3H-diaziren-3-yl)ethyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate: A Derivative of the Stereoselective General Anesthetic Etomidate for Photolabeling Ligand–Gated Ion Channels. Journal of Medicinal Chemistry (2003): 46; 1257-65.