The opening and closing of ligand-gated ion channels (LGICs), which lie in the membranes of nerve cells, regulate information flow throughout the brain. Members of the pentameric LGIC superfamily include nicotinic acetylcholine receptors (nAChR), serotonin-type-3 receptors, gamma-aminobutyric acid type A receptors (GABAR) and glycine receptors. Defects in these channels lead to a variety of neurological diseases and psychiatric disorders. A number of therapeutic drugs, including muscle relaxants, sedative-hypnotics, anti-convulsants, anxiolytics, intravenous and volatile anesthetics, anti-emetics, drugs for nicotine addiction and drugs to treat Alzheimer’s disease target these channels. For these receptors, binding of neurotransmitter in the extracellular ligand-binding domain triggers opening of an intrinsic ion channel more than 50A away in the transmembrane domain of the receptor. Although we know a fair amount about the structure of these receptors, the mechanisms by which the binding of neurotransmitter triggers channel opening and the binding of drugs modulate pLGIC function are relatively unknown.
The major focus of the lab is on understanding the function and structure of the GABAR. GABARs mediate the majority of inhibition in the brain and are modulated by a variety of clinically important drugs, such as benzodiazepines, barbiturates, neurosteroids, anesthetics and anti-convulsants. Furthermore, GABAR mutations have been linked to familial epilepsies, schizophrenia and autism.
Work in the lab is focused on understanding how GABARs and related pLGICs work. First, how does GABA binding trigger the opening of the chloride-selective pore? Second, how does the binding of different drugs such as benzodiazepines, anesthetics, neurosteroids and barbiturates modulate GABAAR function? Third, how do GABAR defects alter receptor function and contribute to disease? Finally, how do neurons control the assembly, trafficking and cell surface expression of GABARs? We are using an array of biochemical, biophysical and electrophysiological approaches including two-electrode voltage clamping and patch-clamping, voltage clamp fluorimetry, rapid-ligand application, disulfide trapping, cysteine cross-linking, structural modeling, protein over-expression and purification, and SDSL-EPR to advance our understanding of how neurotransmitter binding is linked to pLGIC activation and how drug binding is coupled to receptor modulation.
Our work is providing new insights into how neurotransmitters activate pLGICs and how allosteric drugs modulate their activity. A deeper understanding of how these channels work at a molecular level will improve our ability to predict the actions of drugs and ligands that act on these channels, design safer and more effective drugs, develop better therapeutic strategies, and understand the etiology of disease-causing mutations.
Project: Role of Intracellular Domain:
Rapid opening and closing of GABAR channels regulate signaling in the brain. Mechanisms underlying channel open, closed, desensitized transitions remain unclear. GABAR cryo-EM structures provide details about its extracellular and transmembrane domains, but little information exists about its intracellular domain and its role in regulating channel gating. Using GABAR expression in Xenopus laevis oocytes and two-electrode voltage clamping, we are examining if disrupting potential charge interactions between regions in the GABAR intracellular domain alter GABAR gating.
Project: Uncovering Allosteric Pathways:
This project involves previous work done on the b4-b5 linker region of the extracellular domain. This linker region is shown to be critically involved in benzodiazepine (BZD) modulation of the GABA-A-R. We are currently performing mutagenesis studies within this b4-b5 linker region to further understand its role in BZD modulation.
Project:
This project is focused on understanding the role of the putative endogenous benzodiazepine, the diazepam binding inhibitor. This small protein is thought to bind the GABAR in the extracellular benzodiazepine site, and has been shown to act as both a positive and negative allosteric modulator in different regions of the brain. We use two-electrode voltage clamp, whole-cell and patch clamp electrophysiology alongside radioligand binding studies to examine the effects of purified diazepam binding inhibitor on different GABAR subtypes. As we learn more about how this protein affects the GABAR, we strengthen our understanding of the how the brain regulates inhibitory signaling and gain new strategies for designing positive and negative GABAR-modulating drugs.
Project 1:
In this project, we focus on the assembly of GABAAR subunits and their association with accessory proteins during trafficking, translocation and diffusion to extrasynaptic and synaptic sites. Using immunoprecipitation and shRNA techniques, we are studying protein interactions between GABAAR subunits and specific accessory proteins involved in the receptor regulation and functionality.
The lab examines the structure of the GABA-A-R to better understand how receptor structure affects function. Using molecular dynamic simulations and modeling, we are able to visualize how the receptor’s structure may change with drug binding, and also understand different states (closed & open) and conformations the GABA-A receptor may undergo.