An evolutionarily conserved AnkyrinG-dependent motif clusters axonal K2P K+ channels.

The evolution of ion channel clustering at nodes of Ranvier enabled the development of complex vertebrate nervous systems. At mammalian nodes, the K+ leak channels TRAAK and TREK-1 underlie membrane repolarization. Despite the molecular similarities between nodes and the axon initial segment (AIS), TRAAK and TREK-1 are reportedly node-specific, suggesting a unique clustering mechanism. However, we show that TRAAK and TREK-1 are enriched at both nodes and AIS through a common mechanism. We identified a motif near the C-terminus of TRAAK that is necessary and sufficient for its clustering. The motif first evolved among cartilaginous fish. Using AnkyrinG (AnkG) conditional knockout mice, CRISPR/Cas9-mediated disruption of AnkG, co-immunoprecipitation, and surface recruitment assays, we show that TRAAK forms a complex with AnkG and that AnkG is necessary for TRAAK's AIS and nodal clustering. In contrast, TREK-1's clustering requires TRAAK. Our results expand the repertoire of AIS and nodal ion channel clustering mechanisms and emphasize AnkG's central role in assembling excitable domains.