Keith W. Miller, D. Phil.
Vice Chair for Research; Director, Mallinckrodt Pharmacology Lab; Mallinckrodt Professor of Pharmacology in Anesthesia, Harvard Medical School
My research in the Mallinckrodt Molecular Pharmacology Research Unit is concerned with the mechanism of action of general anesthetics with an emphasis on molecular level explanations (1). Research focuses on neuronal ligand-gated ion channels such as the GABA(A) and nicotinic acetylcholine receptors. The overall hypothesis is that general anesthetics both enhance neural inhibition by enhancing the activity of GABA(A) receptors and inhibit neural excitation by inhibiting acetylcholine receptors. The team of investigators involved includes Stuart Forman and Douglas Raines at MGH, Jonathan Cohen at HMS, and Karol Bruzik at University of Illinois, Chicago. Current projects are focused on locating the binding sites of etomidate, propofol and barbiturates on GABA(A) receptors. The strategy is to synthesize analogs of these agents that have all the pharmacological properties of the parent agent but in addition can be induced to covalently attach to amino acid residues in their binding pocket by light activation. Great progress has been made with etomidate (2). The GABA(A)R consists of five fairly homologous subunits arranged approximately symmetrically around a central ion channel. In GABA(A)Rs with the subunit composition ?1?3?1?3?2, etomidate?s two binding sites lie between the alpha and beta subunits in the transmembrane domain of the receptor. Each subunit contributes two of its four membrane?spanning ??helices to the binding site, which is located towards the extracellular end of this structural domain (1, 2). The search for suitable photoactivable etomidate derivatives provided structure activity relationships that led to the development of a short acting etomidate, a project spearheaded by Douglas Raines. Recently, a photoactivable analogs of propofol (3) and a barbiturate have been developed development.
How does binding at these general anesthetic sites alter channel function? In acetylcholine receptors, we have shown that agents bind within the ion channel within a millisecond of acetylcholine opening it. However, other agents bind to a single subunit in a transmembrane site bounded by four helices, and this site is involved in modulating desensitization (4). The detailed mechanisms underlying these actions, and those on the GABA(A)R remains to be attacked.
The development of alcohol derivatives that are photoactivable has enabled us to locate an alcohol?binding site on the neural cell adhesion molecule, L1 (5). This protein was hypothesized by our collaborator, Michael Charness, to be involved in fetal alcohol spectrum disorder because mutations in it result in developmental problems comparable to those induced in children by excessive drinking by pregnant women. The extracellular domain of this 1,257?residue protein is composed of a string of five FnIII and six Ig domains. We photolabeled a residue on Ig1 and another 385 residues away on Ig4. It was hypothesized that the Ig1 and Ig2 units fold back on the Ig4 and Ig3 units forming a shepherd?s crook structure that placed the two photolabeled residues in a single pocket. Subsequent cross-linking experiments have confirmed that these two residues are indeed in close proximity. Thus, once again we find an anesthetic site that is between two structural domains rather than buried within a protein.
List of grant funding
2P01-GM058448-11 (Program Director: Miller) - 09/30/09 - 08/31/14
General Anesthetics Sites on Ligand-Gated Ion Channels
The aims of this PPG is to locate the anesthetic binding sites on the GABA(A), nAChR and 5-HT3 receptors using anesthetic photolabels and define how their occupancy allosterically modulates receptor function using rapid perfusion patch clamp techniques.
1R01-GM087316-01A1 (PI: Raines. Role: Co?Investigator) - 01/01/10 ? 12/31/13
Etomidate Analogues as Safer General Anesthetics
The goal of this project is to develop novel anesthetics that retain etomidate?s beneficial properties, but whose impact on steroid synthesis is greatly reduced.
2R01AA012974-11A1 (PI: Charness. Role: Co?Investigator)
Alcohol and Cell Adhesion
Fetal alcohol spectrum disorder (FASD) is the most common reversible cause of mental retardation. The goal of this research is to expand our knowledge of the structural determinants of the ethanol binding pocket on the neural cell adhesion molecule, L1, in order to facilitate the rational design of ethanol antagonists.
T32 GM007592 (Program Director: Miller) - 07/01/1978 ? 06/30/13
Research Training for Anesthetists
The overall objective is to produce a cadre of anesthesiologists, well trained in research methodology and knowledgeable in their area of expertise, who will have the ability to continue on to independent research careers in problems of importance to anesthesia and the basic sciences upon which it rests.
* Willing to participate as mentor.
Oxford University, D.Phil
Department of Anesthesia, Critical Care and Pain Medicine
55 Fruit Street
Boston, MA 02114