Monitoring Ligand Binding to Purified HCN4 Channel Proteins

Hyperpolarization-activated cyclic nucleotide-gated (HCN 1-4) channels are the molecular determinants of If/Ih, a mixed cation inward current that controls firing automaticity in pacemaker cells. HCN primarily activated by membrane hyperpolarization, and further regulated cAMP binding to cytosolic nucleotide domain (CNBD). Biochemical measurements ligand purified eukaryotic proteins challenging because their low yield expression purification. Starting for an existing assay1, we set protocol detecting HCN4 channel proteins. The same assay was used detect Ivabradine, pharmacologically well-known blockers. is based on possibility monitor stabilizing effect thermal denaturation curve protein. Changes melting temperature protein provide quantitative value measuring ligand-protein interaction. To overcome limit protein, specific signal GFP tag fused HCN4. 1)Emmanuel Nji, Yurie Chatzikyriakidou, Michael Landreh & David Drew. An engineered thermal-shift screen reveals lipid preferences prokaryotic Nature Communications volume 9, Article number: 4253 (2018).