Three 2019 Pilot and Feasibility Grants Awarded

Nathaniel Huebsch PhD, Assistant Professor of Biomedical Engineering

Jianmin Cui, PhD, Professor of Biomedical Engineering

“Optogenetic Toolbox for Controlling Action Potentials in Human iPSC-Derived Cardiomyocytes”

Our immediate aim is to combine our complementary expertise in ion channel biophysics and molecular biology (Cui lab1–4), along with Human Induced Pluripotent Stem Cells (iPSC) and micro-fabrication (Huebsch lab5–7) to develop a toolbox, using existing molecular optogenetics reagents8, to allow us to model exercise-induced arrhythmia in highly defined in vitro models using human iPSC derived cardiomyocytes and Engineered Heart Tissues (EHT; Fig. 1). We plan to leverage this toolbox to model exercise-induced arrhythmias that cause sudden death in patients that carry familial heart diseases, including Long QT Syndrome Type 1 and Arrhythmogenic Cardiomyopathy.

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Peng Yuan, PhD, Assistant Professor of Cell Biology & Physiology

“CIMED mammalian tissue culture facility for large-scale expression of human membrane proteins”

One of the major long-term goals of the Center for the Investigation of Membrane Excitability Diseases (CIMED) is to advance understanding of ion channels and transporters that play essential roles in physiology and disease. It has been well documented that dysfunction of these integral membrane proteins leads to a variety of diseases such as asthma, hypertension, cancer, heart failure, diabetes, chronic pain, etc. Unraveling the inner workings of these membrane proteins are of central importance in biology and will also establish the foundations for rational therapies in medicine. To this end, we need to be able to integrate interdisciplinary approaches ranging from cell biology, physiology, biochemistry, biophysics, and structural biology to obtain detailed molecular understandings of these essential membrane proteins. Knowledge obtained from these studies will facilitate drug development for numerous diseases. In particular, biochemical, biophysical, and structural analyses require large amounts (milligrams) of purified human membrane proteins, which are very often the major bottlenecks hindering our progress in these studies and hence rational development of therapeutics. This application aims to expand the existing capability of eukaryotic (especially human) membrane protein production in CIMED, facilitating studies of various membrane proteins of interest. In CIMED, we have already established excellent large-scale membrane protein production using the bacterial and yeast expression systems, which allowed the characterization of multiple ion channels and transporters. However, we still lack a facility that supports large-scale mammalian cell culture (liters of mammalian cells growing in suspension), which is critical for advancing many potential projects from multiple laboratories affiliated with CIMED. This facility becomes especially critical with our growing interest in eukaryotic or even human membrane proteins. To this end, we definitely need to set up an efficient large-scale mammalian cell culture facility for the study of human membrane proteins of interest, which will potentially benefit research in multiple labs affiliated with CIMED.

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Wayland Cheng, MD, PhD, Assistant Professor of Anesthesiology

“Characterizing Lipid and Anesthetic Interactions with Pentameric Ligand Gated Ion Channels using Native Mass Spectrometry”

The overarching goal of this work is to understand the mechanism by which lipids and hydrophobic small molecules such as anesthetics bind to and modulate the structure and function pentameric ligand-gated ion channels (pLGICs). The objective of this pilot proposal was to optimize native MS and ion mobility techniques to study individual lipid and drug binding events to pLGICs, and to examine the effect of binding at different stoichiometries on channel stability and unfolding. During the first year of funding, I made significant progress in developing these techniques to study phospholipid binding to the pLGIC, ELIC. In addition, this pilot grant was criticized by reviewers for not developing techniques complementary to native MS, which would be necessary to secure an external grant. In response, I have also developed the capability to study ELIC channel function reconstituted in model membranes by patch-clamping giant liposomes. In addition, I have reconstituted ELIC in lipid nanodiscs, and made some progress using cryo-electron microscopy to obtain high resolution structures in different lipid environments. Lastly, I am in the process of developing collaborations to model lipid binding in pLGICs using molecular dynamics simulations. In addition to showing feasibility, this work is yielding novel insights into the mechanism of lipid modulation of pLGICs.