Ion channel inhibition by targeted recruitment of NEDD4-2 with divalent nanobodies

ABSTRACT: Targeted protein degradation or downregulation (TPD/TPDR) mediated by induced recruitment of an endogenous E3 ubiquitin ligase to a substrate is a disruptive paradigm for developing therapeutics. However, <2% of ~600 E3 ligases have been exploited for this modality, and efficacy for multi-subunit membrane ion channel complexes has not been demonstrated. The HECT E3 ligase NEDD4-2 regulates myriad ion channels, but it’s utility for TPD/TPDR is unexplored and uncertain due to complex intramolecular and posttranslational regulation mechanisms. We identified a nanobody that binds NEDD4-2 HECT domain strongly without disrupting sites critical for catalysis as revealed by a cryo-electron microscopy reconstruction and in vitro ubiquitination assays. Recruiting NEDD4-2 to diverse ion channels with distinct topologies (high voltage-activated CaV2.2 calcium channels; voltage-gated potassium channel, KCNQ1; and the epithelial Na+ channel, ENaC, with a clinically relevant Liddle syndrome channelopathy mutation) using divalent nanobodies (DiVas) strongly suppressed their cell surface density and function, even when there was no frank degradation. Global proteomics indicated DiVa recruitment of endogenous NEDD4-2 to a test ion channel, KCNQ1-YFP, displayed dramatically lower off-target effects compared to NEDD4-2 overexpression. The results establish utility of induced recruitment of NEDD4-2 for TPD/TPDR, validate multi-subunit ion channels as susceptible to this modality, and introduce DiVas as a general method to generate ion channel inhibitors.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Henry Colecraft  

LAB HEAD: Henry Colecraft

PROVIDER: PXD069051 | Pride | 2026-01-19

REPOSITORIES: Pride