Disease-associated Kv1.3 variants are energy compromised with impaired nascent chain folding.

Human Kv1.3, encoded by , is expressed in neuronal and immune cells. Its impaired expression or function produces chronic inflammatory disease and autoimmune disorders, the severity of which correlates with Kv1.3 protein expression. The intersubunit recognition domain, T1, at the cytosolic N-terminus of Kv1.3, acquires secondary, tertiary, and quaternary structures during early biogenesis while the nascent protein is attached to the ribosome and/or the ER membrane. In this study, we ask whether native gene variants in T1 are associated with human disease and whether they manifest early-stage folding defects, energetic instabilities, and conformational distortion of subunits. We use three approaches: first, the unbiased "genome-first" approach to determine phenotype associations of specific rare variants. Second, we use biochemical assays to assess early-stage tertiary and quaternary folding and membrane association of these variants during early biogenesis. Third, we use all-atom molecular dynamics simulations of the T1 tetramer to assess structural macroscopic and energetic stability differences between wildtype (WT) Kv1.3 and a single-point variant, R114G. Measured folding probabilities and membrane associations are dramatically reduced in several of the native variants compared to WT. Simulations strikingly show that the R114G variant produces more energetically unstable and dynamic T1 domains, concomitant with tertiary unwinding and impaired formation of symmetrical tetramers. Our findings identify molecular mechanisms by which rare variants influence channel assembly, potentially contributing to diverse clinical phenotypes underlying human disease.