Yaoli Pu with Bin Ye and Professor Jonathan Makielski

UW-Madison Department of Cardiology – Ion Channels

Beating at a pace of 100,000 times a day, the human heart’s specialized atrial and ventricular cells (“working myocytes”) rely on an external trigger – the sinoatrial node (SAN), to keep the cardiac rhythm. Unlike normal myocytes (which remain at a stable voltage level until a stimulus appears), the SAN myocytes have a unique current important for pace-making. After termination of a signal, the channels slowly depolarize up to threshold for another action potential, causing spontaneous firing of cells. Hyperpolarization-activated, cation nonselective (HCN) channels are the molecular components of pacemaker channels and have four isoforms comprising of four subunits. We focused mainly on HCN4 channels. In order to study the protein, a marker needs to be placed in the gene encoding for HCN4 channels so that we have an “experimental” version that is easily identified. However, inserting a mutation (~ 15 bp) was difficult because the gene is G-C reach and therefore hard to separate in mutagenesis PCR. After using several strategies, we were able to insert the marker into the gene. The next step would be to transfact the genes into human kidney cells and compare the voltage of the “experimental” HCN4 channel to wildtype HCN4 channels to check the effect of the marker mutation.