State Dependent Photo-Crosslinking of the IKS Channel Complex Demonstrates Movement of KCNE1 at Pre-Opening Membrane Potentials

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 the dynamics of 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. Characterization of the pBpa-incorporated channel complex revealed a 9 mV left shift in V0.5 of activation compared to wild type. To evaluate the channel's activation pathway, cells were held for 2s at a range of non-activating potentials (−110 - −30 mV) followed by a 4s activation step to +60 mV. Increasing the holding potential progressively reduced activation time confirming multiple closed-states. Crosslinking was induced for each non-activating potential by repeatedly applying a 300 ms flash of UV light at the end of the 2s hold followed by a 4s activation step to +60 mV. Analysis of the change in peak current vs. cumulative UV-exposure revealed a rapid decrease compared to wild type channels indicating the permanent trapping of closed channels. The greatest rates of crosslinking were found at the most hyperpolarized holding potentials but no significant change in rate was observed above −70 mV, indicating that KCNE1 has moved outside the pBpa crosslinking radius as the channels progress through the activation pathway. This initial movement of KCNE1 suggests that inhibition of KCNQ1 occurs in a closed-state closer to the open-state in the activation pathway.