Research Interest

We study membrane proteins. The activity of virtually every cell is regulated by extracellular signals, such as neurotransmitters, hormones, and sensory stimuli. These signals are transmitted into the cell interior via membrane receptor proteins. Understanding how these membrane proteins mediate signal transmission and transduction is our primary research interest. In particular, we are interested in the structure and function relationship, the kinetic and molecular mechanism of protein function by protein-protein and protein-drug interactions. We also attempt to develop better inhibitor/potentiators to regulate membrane protein functions. In the long term, we hope that our research will provide not only insight into the mechanisms of action of these molecular machines, but also clues for design of molecular devices which can be used (i) for studying signal transduction pathways and (ii) as diagnostic/detection tools for disease treatment. We use an interdisciplinary approach in our research, including rapid kinetic techniques suitable for membrane proteins, biochemical and biophysical chemistry, molecular biology, electrophysiology and neuroscience.

Current Research Projects

Our current research centers on glutamate ion channel receptors. These receptors mediate synaptic neurotransmission and are indispensable in the brain activity, such as memory and learning. Excessive receptor activation, however, leads to neurodegeneration. Inhibitors of glutamate receptors are thus promising agents to treat neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Upon binding to glutamate, the glutamate receptor rapidly changes its conformation and opens its ion channel pore to allow small cations such as sodium ions to flow across the cellular membrane, thus transmitting an electrical signal between neurons. Because the receptor channel opens in the microsecond second (ms) time region and desensitizes even in the millisecond (ms) time domain, a rapid kinetic technique must be used that not only has a sufficient time resolution but also is suitable for studying these channel proteins embedded in membrane. We use a laser-pulse photolysis technique, which permits glutamate to be liberated from γ-O-(α-carboxy-2-nitrobenzyl)glutamate (or caged glutamate) with a time constant of ~30 µs. This technique, combined with electrophysiological recordings, serves as a unique functional tool so that we can investigate the mechanism of channel formation, inhibition and regulation within the µs-to-ms time domain. The following are some projects we are working on.

1. Kinetic Mechanism of Channel Opening

We are characterizing the kinetic mechanism of receptor channel opening using the laser-pulse photolysis technique. Knowing the kinetic mechanism of channel opening enables a more accurate prediction of the time course of the open channel as a function of glutamate concentration, which determines the transmembrane voltage change and in turn controls synaptic neurotransmission. Furthermore, characterizing the channel-opening rate constants is required to define the integration of nerve impulses that arrive at a synapse or that are generated at a synapse from different glutamate receptors, yet responding to the same chemical signal, i.e., glutamate.

2. Structure-Function Relationship

In this study, we are particularly interested in the effect of structural variations (either by nature such as RNA splicing and editing or by genetic engineering) on the receptor function in the µs-to-ms time domain. For instance, a single leucine-to-tyrosine substitution in AMPA-type glutamate receptors renders the homomeric channel virtually non-desensitizing. In contrast, the wild type receptor desensitizes rapidly. Our study shows that the mutant closes its channel much more slowly. Thus, the mutation is primarily responsible for turning the receptor into a very stable open state.

3. Mechanism of Inhibition

Excessive activation of glutamate receptors, such as AMPA type, is known to induce calcium-dependent excitotoxicity that leads to neurodegeneration. Therefore, developing specific receptor inhibitors has been a long pursued therapeutic strategy. We are investigating how synthetic inhibitors block the receptor channel opening in the µs-to-ms time region. We are also using these compounds as mechanistic and structural probes to investigate the location and properties of the sites of interaction between inhibitors and receptors.

4. Developing Nucleic Acid-Based Drug Candidates for Potential Treatment of Neurodegenerative Diseases

Unlike traditional chemical synthesis, we are using a molecular biology approach called SELEX (systematic evolution of ligands by exponential enrichment) and are developing a new class of inhibitors from combinatorial nucleic acid libraries. Some inhibitors we identified have both nanomolar affinity and novel mechanism of action. They are unique templates for design of drugs and diagnostic reagents with higher affinity and selectivity.

5. Structural Studies

We are also using NMR to determine the structures of chemical inhibitors and nucleic acid inhibitors in the absence and presence of the S1S2 AMPA receptors. The S1S2 receptor is a partial receptor protein containing the extracellular binding domain. These studies are coupled with biochemical characterization of the interface between the protein and the small molecules using techniques such as footprinting. Our goal is to develop more effective and powerful small molecules for regulating receptor function.

Examples of our studies and results can be found in the publication list.