Early-stage determinants of T1-S1 conformations in Kv1.3 channels

Early-stage biogenesis of voltage-gated potassium channels (Kv) remains remarkably understudied yet is key to defining co- and post-translational acquisition of Kv secondary, tertiary, and quaternary conformations. Thus, we have studied nascent folding events and their determinants in Kv1.3, a tetrameric ion channel highly expressed in nerve and immune cells. We explore how folding of T1, an intersubunit recognition domain that ensures correct isoform assembly, is modulated by molecular determinants in the Kv subunit. Specifically, we focus on the T1-S1 linker and its highly conserved C-terminal sequence, S0. Using pegylation, a mass-tagging strategy, inter- and intrasubunit crosslinking, we define the molecular determinants of T1-S1 linker accessibility and location. Our findings show that i) dynamic protein-lipid and protein-protein linker interfaces exist, ii) the presence of a T1 domain and its conformation (monomer versus tetramer) impact linker properties, iii) helical formation of S0 occurs in early biogenic stages and does not require the presence of membranes, iv) a core recognition domain (T2) increases T1 dimerization efficiency and promotes tetramer formation, and v) as few as 12 native linker residues enable T1 tetramerization. These findings differ from canonical models and suggest that domain plasticity for Kv1.3 contributes to biogenesis and may underlie domain-domain communication in Kv function.