Overview

Edward Mallinckrodt, Professor of Pharmacology

Research Areas

• Action of alcohol binding sites on the neural adhesion molecule, L1, responsible for fetal alcohol spectrum disorder
• Location and allosteric action of anesthetic sites on ligand-gated ion channels such as GABAA receptors
• Molecular mechanisms of action of general anesthetics

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 transmembrane domain of four helices, and an intracellular domain. Ongoing research focuses on locating the various binding sites for different structural classes of general anesthetics on GABAA receptors. To achieve this aim, we have developed 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, barbiturates and steroids. The synaptic GABAA receptor’s five subunits are arranged around the ion channel in the order γβαβα (clockwise viewed from the extracellular side). Working with collaborators in a National Institute for General Medical Sciences funded program project grant, it has been determined that etomidate has two identical binding sites in the transmembrane domain between β and α subunits (the β – α interfaces). A newly developed barbiturate general anesthetic binds mainly at the single γ – β subunit interface. An existing knockin mouse that had been engineered to be insensitive to etomidate, is normally sensitive to the new barbiturate, consistent with the different binding sites we found in vitro. We also developed a photoactivable convulsant barbiturate and have found a third site on GABAARs. The current focus is on steroid anesthetics.

Research in my laboratory is now beginning to focus on how these binding sites allosterically affect GABAAR function. Why do they stabilize some functional conformations rather than others? To study this, we attached spectroscopic reporter groups close to the binding sites and examine how their signal changes as a function of the receptor‘s conformation. Specifically, we are using site-directed spin labeling with pulsed electron paramagnetic resonance to answer these questions.

Prenatal ethanol exposure results in diverse birth defects and neurobehavioral abnormalities (FASD) 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.

Publications

Chiara, DC, Jayakar, SS, Zhou, X, Zhang, X, Savechenkov, PY, Bruzik, KS, K.W. Miller, KW, Cohen, JB. Specificity of intersubunit general anesthetic binding sites in the transmembrane domain of the human alpha1beta3gamma2 GABAA receptor. The Journal of Biological Chemistry 2013: 288:19343-19357. PMID: 23677991; PMCID: 3707639.

Jayakar SS, Zhou X, Chiara DC, Dostalova Z, Savechenkov PY, Bruzik KS, Dailey WP, Miller KW, Eckenhoff RG, Cohen JB. Multiple Propofol-binding Sites in a γ-Aminobutyric Acid Type A Receptor (GABAAR) Identified Using a Photoreactive Propofol Analog. J Biol Chem. 2014; 289: 27456-68. PMID: 25086038; PMCID: PMC4183786

Jayakar SS, Zhou X, Chiara DC, Savechenkov PY, Chiara DC, Desai R, Bruzik KS, Miller KW, Cohen JB. Positive and Negative Allosteric Modulation of an α1β3γ2 γ-Aminobutyric acid Type A (GABAA) Receptor by Binding to a Site in the Transmembrane Domain at the γ+ – β– Interface*. J Biol. Chem. 2015; 290: 23432–23446. PMID: 26229099. PMCID: PMC4645599.

Desai R, Savechenkov PY, Zolkowska D, Ge R, Rogawski RA, Karol S. Bruzik KS, Forman SA, Raines DE, Miller KW. Contrasting actions of a convulsant barbiturate and its anti-convulsant enantiomer on the α1β3γ2L GABAA receptor account for their distinct in vivo effects. J Physiol. 2015; 22: 4943–4961. PMID: 26378885 PMCID: PMC4650410.

Forman SA, Miller KW. Mapping General Anesthetic Sites in Heteromeric Gamma-Aminobutyric Acid Type A Receptors Reveals Potential For Targeting Receptor Subtypes. Anesthesia and Analgesia. 2016: in press.