Massachusetts General Hospital

Edward Mallinckrodt Professor of Pharmacology

Research Areas

• Molecular mechanisms of action of general anesthetics
• Location and function of anesthetics sites on ligand-gated ion channels such as GABAA receptors.
• Location of alcohol sites on the neural adhesion molecule, L1, responsible for fetal alcohol spectrum disorder.
• Interaction of etomidate derivatives with β–hydroxylase (CYP 11B)

Description of Research

My laboratory focuses on elucidating the molecular mechanisms by which general anesthetics act to produce anesthesia and its side effects. Our overall hypothesis is that anesthesia is caused by action on neuronal ligand-gated ion channels. Anesthesia may result from general anesthetics enhancing the action of inhibitory receptors (GABAA or Glycine receptors), inhibiting the action of excitatory receptors (nicotinic, 5HT3A or glutamate receptors) or a mixture of both actions. Of these receptors, the Cys loop family is our current focus. They consist of five roughly homologous subunits arranged centro–symmetrically around a central ion–conducting channel. Each subunit consists of an extra cellular domain that binds the agonist, a domain of four transmembrane helices, and an intracellular domain. Currently, we are focusing on locating the binding sites for general anesthetics on these receptors. To achieve this aim, we develop new general anesthetics that have the property of being stable under physiological conditions but becoming reactive when they are stimulated with light, at which time they can covalently attach to amino acid residues in their binding sites. Classes of anesthetic under study include etomidate, propofol and barbiturates. On nicotinic receptors, anesthetics mostly bind in the lumen of the ion channel, but there is also a site on one subunit located within the bundle of four
transmembrane helices. Both these sites have low affinity in the unstimulated resting state of the receptor, but are occupied by anesthetics within milliseconds of the agonist opening the ion channel.

We are now focused on the enhancing action of general anesthetics on the GABAA receptor. We have developed a photoactivable analog of etomidate that causes anesthesia in vivo with the same potency and stereoselectivity as etomidate. The most common GABAA receptor in the brain has the two each of α− and β−subunits and one γ−subunit arranges in the order αβαβγ clockwise around the ion channel. We find that etomidate has two identical binding sites in the transmembrane domain between the α− and β−subunits. This intersubunit site was unexpected. We are now studying barbiturates and find that they have low affinity for the etomidate site.

Prenatal ethanol exposure results in diverse birth defects and neurobehavioral abnormalities in up to 1 per 100 live births. In severe cases, it is characterized by growth deficiency, neurological abnormalities, and facial malformations. In collaboration with the laboratory of Michael Charness, we are testing the hypothesis that ethanol causes FASD in part by disrupting the function of the L1 cell adhesion molecule. We have photolabeled L1 with analogs of alcohols. The two residues photolabeled are far apart in the sequence, but by building a homology model of L1, we were able to show that the residues are located close to each other in a single binding pocket between the Ig1 and Ig4 domains of L1. After the two residues were mutated to cysteines, cell adhesion experiments revealed they could be cross–linked. Further mutations changed the size of the pocket causing a shift in the structure activity relationship of alcohols.

Selected Publications

• Forman SA, Miller KW. Anesthetic Sites and Allosteric Mechanisms of Action on Cys-loop Ligand-gated Ion Channels. Can J Anesthesiol. 2011; 58:191-205. PMCID: PMC3108180.
• Savechenkov, PY, Zhang, X, Chiara, DC, Stewart, DS, Ge, R, Zhou, X, Raines, DE, Cohen , JB, Forman, SA, Miller, KW, Bruzik, KS. Allyl m-Trifluoromethyldiazirine Mephobarbital: An Unusually Potent Enantioselective and Photoreactive Barbiturate General Anesthetic. Journal of Medicinal Chemistry 2012: 55; 6554−6565. PMID: 22734650.
• Chiara, DC, Dostalova, Z, Jayakar, SS, Zhou, X, Miller, KW, and Cohen, JB. Mapping General Anesthetic Binding Site(s) in Human alpha1beta3 gamma-Aminobutyric Acid Type A Receptors with [(3)H]TDBzl-Etomidate, a Photoreactive Etomidate Analogue. Biochemistry, 2012. 51: 836-47. PMCID: 3274767. PMID 22243422.
• Stewart DS, Savechenkov PY, Dostalova Z, Chiara DC, Ge R, Raines DE, Cohen JB, Forman SA, Bruzik KS, Miller KW. p-(4-Azipentyl)-propofol: A Potent Photoreactive General Anesthetic Derivative of Propofol. Journal of Medicinal Chemistry 2011: 54: 8124–8135. PMID 22029276.
• Dou X, Menkari C, Shanmugasundararaj S, Miller KW, Michael E. Charness ME. Two alcohol binding residues interact across a domain interface of the L1 neural cell adhesion molecule and regulate cell adhesion. J. Biol. Chem. 2011: 286: 16131-9. PMCID: 3091222. PMID 21367865

Anesthesia, Critical Care and Pain Medicine

Phone: 617-726-3030

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