Studying the Role of SUMO in the Interaction between HCN2 Channel and its Auxiliary Subunit TRIP8b Using Pull-down Assay

Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels form an integral part of the plasma membrane, existing as tetramers and enclosing ion conducting pores. Ion channels are often regulated by auxiliary subunits and one such protein interacting with the HCN channel is TPR-containing Rab8b interacting protein (TRIP8b). TRIP8b is known to play a crucial role in controlling voltage dependence of HCN channel activation, channel trafficking and surface expression. Small Ubiquitin-like Modifier (SUMO) is a peptide that can be conjugated to a lysine residue on a target protein and modify protein- protein interactions. HCN2 channel can be SUMOylated and possible SUMOylation sites on TRIP8b exist. SUMOylation at these sites may strengthen or weaken the association between these proteins. In this study we use Pull-down assay technique to determine whether SUMO influences this interaction. By altering the baseline SUMOylation level, changes in the degree of HCN channel protein pulled down by TRIP8b is analyzed.