PROJECT SUMMARY As the site of action potential initiation, the axon initial segment (AIS) contains high densities of voltage-gated sodium (Nav) and potassium (Kv) channels that generate and shape the action potential. Kv1 channels are a prominent type of ion channel at the AIS that modulate action potential waveform. Despite their functional importance, the mechanisms responsible for the localization of these channels at the AIS are poorly understood. In contrast, the recruitment of other ion channels to the AIS is well established and depends on interaction with the AIS scaffold protein, AnkyrinG (AnkG). Nav1 and Kv7 channels directly interact with AnkG through a conserved binding motif that Kv1 channels lack. However, unlike Nav1 and Kv7 channels, Kv1 channels contain PDZ binding motifs, which permit interactions with PDZ domain containing proteins. Kv1 channels have been shown to interact with the PDZ domain-containing scaffold protein PSD93 that is highly enriched at the AIS. Although in vitro knockdown studies found that PSD93 is required for Kv1 channel clustering at the AIS, subsequent in vivo knockout studies demonstrated that PSD93 is dispensable, suggesting that other mechanisms can cluster Kv1 channels at the AIS. Previous work also suggests that AnkG is required for AIS Kv1 channel clustering, but the link between AnkG and Kv1 channels is unknown and may be indirect. Thus, the mechanisms underlying Kv1 channel localization at the AIS are unclear. Our lab recently identified a new AIS scaffold protein, SCRIB, that directly binds AnkG. SCRIB contains multiple PDZ domains and may constitute a novel mechanism for clustering Kv1 channels at the AIS. However, the function of SCRIB at the AIS has not been investigated. The objective of this proposal is to define the molecular mechanisms responsible for clustering Kv1 channels at the AIS. Aim 1 will use AAV- and CRISPR-based knockouts, conditional knockout mouse models, and immunostaining to determine if SCRIB, PSD93, and AnkG are necessary for AIS Kv1 channel clustering in vitro and in vivo. Aim 2 will use co-immunoprecipitations and surface clustering assays in heterologous cells to determine the molecular interactions between Kv1 channels and the AIS scaffold proteins SCRIB, PSD93, and AnkG. The proposed experiments will establish a model for how Kv1 channels are localized at the AIS. The Rasband lab has extensive experience elucidating the molecular mechanisms underlying AIS assembly. Thus, the proposed experiments are designed to take advantage of this expertise and the many tools and reagents available in the Rasband lab. This research project will also provide a robust training experience for my development as a molecular neurobiologist. Through the completion of the proposed aims, I will advance the research skills, critical thinking skills, scientific communication skills, and mentorship skills that I will need for my future career as an independent investigator.