State-Dependent Crosslinking in IKS Demonstrates a Closed-State Interaction between KCNE1 at F57 and KCNQ1 that Inhibits Channel Opening

Abstract

The slow delayed rectifier current (IKs) is a key repolarizing potassium current in the cardiac action potential. IKs is composed of KCNQ1 which forms the tetrameric voltage gated pore subunit and KCNE1, a single transmembrane domain accessory subunit, proposed to reside in the channel's exterior cleft. KCNE1 imposes a dramatic regulation on KCNQ1, significantly delaying opening compared to the unchaperoned channel. Here, we have investigated this interaction using the UV-crosslinking unnatural amino acid, p-benzoyl-L-phenyl alanine (pBpa). pBpa was genetically incorporated into KCNE1 at residue F57 in the transmembrane domain using the amber stop codon (TAG) suppression system. Successful incorporation into KCNE1-F57TAG was confirmed by a lack of functional IKs current in transfected cells not supplemented with pBpa. Characterization of the pBpa-incorporated channel complex revealed a right shifted V0.5 of activation compared to wild-type (+27 mV vs. +14 mV). Channels were UV-irradiated in the closed state by applying a 300 ms light pulse at −90 mV followed by a 4s activation step (+60 mV). A diary plot of the peak current vs. UV-exposure with repeated exposure revealed a rapid decrease in available current compared to UV-treated wild-type channels. This indicates the permanent closure of channels by crosslinking. Application of UV at the end of a 4s activation step (+60 mV) produced an immediate downward deflection in current and resulted in a slower rate of channel crosslinking compared to the closed-state UV treatment. This suggests that KCNE1 can revisit a closed-state orientation during channel activation and is then trapped in the closed-state by the covalent crosslink. These findings provide new insight into the interactions that regulate the IKs channel complex.