Developing a Model Heterologous Cell Line Expressing Human Cardiac ATP-Sensitive Potassium Channels
Department or Program
Primary Wellesley Thesis Advisor
Cardiomyocytes can be exposed to a variety of stressful conditions. Of particular interest is the response of these cells to hypoxic conditions. In humans, the heart can become hypoxic from underlying physical conditions, such as atherosclerosis leading to ischemia. ATP-sensitive potassium (KATP) channels have been observed to protect cardiomyocytes from damage under low-oxygen conditions. Without sufficient oxygen, mitochondria cannot perform oxidative phosphorylation to convert ADP to ATP. When the ratio of ATP to ADP becomes low enough, KATP channels open, causing potassium ions to flow out of the cell. This efflux of potassium is thought to protect cardiomyocytes from low oxygen conditions by 1) minimizing ATP consumption and by 2) preserving mitochondrial membrane integrity by reducing the influx of calcium ions, which can trigger of apoptosis. The goal of this project is to create a heterologous model for studying the function of KATP channels. Specifically, the focus is to create a stable cell line expressing both subunits of KATP channels: the pore-forming subunit Kir6.2 (encoded by the gene KCNJ11) and the regulatory subunit SUR2A (ABCC9). Here, the Kir6.2 subunit has been transfected into Chinese Hamster Ovary and Human Embryonic Kidney cells. Work is ongoing to improve the efficiencies of these transfections, to detect the expression of this channel on immunoblots, and to establish a stable cell line expressing this subunit alone. With the regulatory subunit, I have subcloned ABCC9 cDNA from a holding vector into a pIRES vector that also expresses a fluorescent reporter protein and mutagenized the ABCC9 sequence to be consistent with the most recent entry in NCBI. In the future, a cell line co-transfected with both subunits should express and form fully-functional cardiac KATP channels. With this model heterologous cell line, the Cameron and Darling Laboratories can begin investigating how the trafficking and activity of these channels change under hypoxic conditions in vitro.