CURF Introduction – There’s traffic in your cells too

Hello! My name is Katie Oppenheimer. I am a Junior majoring in microbiology. I’m from Portland, Oregon. Coming from Oregon, I love to spend time in nature, especially backpacking and skiing. I conduct research in the O’Donnell Lab. The O’Donnell lab studies a-arrestins. These protein are adaptors involved in intracellular protein trafficking, acting as a “bridge” between the membrane protein and the ubiquitin ligase Rsp5. As the environment is constantly changing, a cell must be able to adapt in order to survive. In many cases, the cell does this by altering the composition of the plasma membrane. My project focuses specifically on elucidating the role of various potential trafficking regulators in a-arrestin mediated trafficking of the mammalian potassium channel Kir2.1. Kir2.1 is an inward rectifying potassium channel primarily expressed in the heart, skeletal muscle and neurons. As a transmembrane channel located at the plasma membrane, Kir2.1 plays a significant role in maintaining potassium homeostasis. The control of potassium and calcium ions flux is critical to driving heart contraction. Defects in the movement of ions across the cell membrane result in cardiac arrhythmias and irregular heartbeats. Several Kir2.1 mutations that lead to disease are linked to dysfunctional trafficking of the channel to the plasma membrane, and previous work in the O’Donnell lab has identified three a-arrestins – Aly1, Aly2, and Ldb19 – to be involved in Kir2.1 trafficking.

With this connection to cellular trafficking and arrestins established, I am working to identify regulators of this trafficking, and through that, start to build an understanding of the intracellular pathways that a-arrestins use to traffic Kir2.1. To do this, I am using serial dilution assays and Fluorescent Activated Protein (FAP) imaging technology to evaluate the changes in the amount of Kir2.1 at the plasma membrane. FAP is a powerful imaging technology. It is comprised of two parts: a single chain antibody, which is fused to Kir2.1, and a malachite-green derived dye added prior to imaging. Separate, neither the antibody nor the dye fluoresces, but when the two are bound, there is a 20,000-fold increase in fluorescence. There are two types of dye that can be used. One is membrane – permeant, meaning it will diffuse through the plasma membrane and into the cell, binding to all the antibodies fused to Kir2.1 located on the exterior and interior of the cell. This is valuable if you want to determine the total Kir2.1 of a cell. I am interested, however, only in the portion of Kir2.1 at the plasma membrane (about 10% of total Kir2.1in a cell). Consequently, I use the in membrane-impermeant dye, which will only bind to Kir2.1 on the exterior of the cell, to visualize the small pool of Kir2.1 at the plasma membrane.  

Defining the regulators of this form of intracellular trafficking will improve our understanding of the molecular mechanisms governing how Kir2.1 is localized to maintain K+ homeostasis and cardiac function. With this knowledge, improvement can be made in the treatment of diseases caused by improper Kir2.1 trafficking and protein trafficking-related diseases in general.

In terms of my professional goals, I plan to pursue a PhD, with the long-term goal of becoming a professor and conducting research. This fellowship will allow me to focus on my research and improve my ability to communicate the key points of my research.

  1. Harrell, M.D., Harbi, S., Hoffman, J.F., Zavadil, J. and Coetzee, W.A. (2007) Large-scale analysis of ion channel gene expression in the mouse heart during perinatal development. Physiol Genomics, 28, 273-283.
  2. Hager, N.A., Krasowski, C.J., Mackie, T.D., Kolb, A.R., Needham, P.G., Augustine, A.A., Dempsey, A., Szent-Gyorgyi, C., Bruchez, M.P., Bain, D.J. et al. (2018) Select alpha-arrestins control cell-surface abundance of the mammalian Kir2.1 potassium channel in a yeast model. J Biol Chem, 293, 11006-11021.
  3. Szent-Gyorgyi, C., Stanfield, R.L., Andreko, S., Dempsey, A., Ahmed, M., Capek, S., Waggoner, A.S., Wilson, I.A. and Bruchez, M.P. (2013) Malachite green mediates homodimerization of antibody VL domains to form a fluorescent ternary complex with singular symmetric interfaces. J Mol Biol, 425, 4595-4613.
Hiking in Germany!

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