Dynamic Subunit Stoichiometry of Kv4.3-KChIP4A Channel Complexes Visualized by Single-Molecule Subunit Counting

Abstract

Auxiliary Kv channel-interacting proteins 1-4 (KChIPs1-4) co-assemble with pore-forming Kv4 α-subunits to underlie somatodendritic subthreshold A-type current that regulates neuronal excitability. It has been hypothesized that different KChIPs can competitively bind to Kv4 α-subunit to form dynamic channel complexes that can exhibit distinct biophysical properties for modulation of neural function. To test this hypothesis, we utilized single molecule subunit counting to investigate whether different isoforms of auxiliary KChIPs such as KChIP4a and KChIP4bl can compete for binding of Kv4.3 to co-assemble hetero-multimeric channel complexes. Single-molecule imaging subunit counting revealed that the number of KChIP4 proteins in Kv4.3-KChIP channel complexes can vary depending on KChIP4 expression level. Increasing amount of KChIP4bl gradually reduces the bleaching steps of GFP for KChIP4a proteins and vice-versa. To further demonstrate Kv4 gating affected by different KChIP4 subunit stoichiometry, we generated two tandem constructs to mimic the situations of KChIP4a half-occupied channel complexes (KChIP4a-2xKv4.3) and KChIP4a saturated channel complexes (KChIP4a-Kv4.3) expressed in Xenopus oocytes. Gating kinetics of KChIP4a-2xKv4.3 co-expressed with KChIP4bl (to mimic channel complex like Kv4.3:KChIP4a:KChIP4bl with the ratio of 4:2:2) shows that both KChIP4a and KChIP4bl can simultaneously modulate the function of channel complexes upon co-assembly. The significance of dynamic channel complexes was further investigated in hippocampal neurons from kainic acid seizure model in rats by detecting a shift of expression profile from KChIP4bl to KChIP4a. Our preliminary findings demonstrate that auxiliary KChIPs can hetero-assemble with Kv4 in a competitive manner to form hetero-multimeric Kv4-KChIP4 complexes that are biophysically distinct and dynamically regulated under pathological conditions.