BPS2026 – Exploring the interaction between Kv2.1 and AMIGO using computational and experimental approaches

Protein-protein interactions (PPIs) play an important role in many biological processes, including regulation and modulation of ion channel activity by auxiliary partners. The Kv2.1 voltage-activated potassium (Kv) channel is a delayed-rectifier Kv channel in the central nervous system where it is critical for regulating intrinsic neuronal excitability. Kv2 channels are modulated by amphoteric-induced gene and open reading frame (AMIGO) neuronal adhesion proteins; in particular, there are three AMIGO proteins that shift the activation of the channel to more hyperpolarized potentials. As well, AMIGO2 specifically slows inactivation. Through a combination of computational and experimental techniques, we explore the structural basis of the interaction between the AMIGO family and Kv2.1 and how mutations in the predicted interactions may lead to functional changes in Kv2.1’s electrophysiological properties. AlphaFold2 models of the Kv2.1/AMIGO complexes propose two possible positionings of the AMIGO transmembrane helix. The most frequent structures with AMIGO in a plausible location are those with two AMIGO chains interacting with Kv2.1. These structures show the two chains as a dimer with both AMIGO transmembrane helices in the transmembrane region near the Kv2.1 voltage sensors. With these predictions, we identify contacts between the extracellular end of the AMIGO transmembrane helix with S4 of Kv2.1, potentially involved in activation of the channel, and the intracellular end with a region of Kv2.1 known to be involved with regulating inactivation of the channel. We use two electrode voltage clamp (TEVC) to explore each AMIGO’s functional regulation of Kv2.1 and the effects of mutations within the predicted interaction sites. Through AlphaFold predictions and experimental follow-up, we will identify potential interactions between Kv2.1 and AMIGO which will give insight on the channel’s mechanisms of activation and inactivation as well as the regulation mechanism of the AMIGO family.