Insertion of Crosslinkable Amino Acids into the IKS Channel Complex Demonstrates a Variable KCNQ1:KCNE1 Stoichiometry of up to 4:4

Abstract

The slow delayed rectifier current (IKs) provides repolarizing potassium current during the cardiac action potential. It is composed of KCNQ1, which forms the tetrameric voltage-gated ion channel, and KCNE1, a single transmembrane domain β-subunit. KCNE1 resides in the channels’ exterior clefts and dramatically delays opening. The stoichiometry between the α and β-subunits in the channel complex remains controversial. Several studies have reported either a strict ratio of 2 KCNE1: 4 KCNQ1 or a variable ratio up to 4:4. Here, we aimed to address this issue using IKs fusion proteins, where KCNE1 was linked to one (EQ) or two KCNQ1 subunits (EQQ), to give rise to compulsory 4:4 or 2:4 stoichiometries. Whole cell and single channel characterization of EQQ in mammalian cells demonstrated a hyperpolarized V1/2 of activation, reduced conductance and shorter first latency of opening compared to EQ. All of these differences were abolished by co-expression of EQQ with KCNE1-GFP. To confirm that these additional subunits can be integrated into the complex, the UV-crosslinking unnatural amino acid, p-benzoyl-L-phenylalanine (Bpa), was genetically incorporated into KCNE1-GFP at residue F57 using the amber stop codon (TAG) suppression system. Application of UV light to KCNQ1 + F75Bpa KCNE1-GFP complexes held at −90 mV, trapped channels in the closed state. The same UV-treatment of F57Bpa KCNE1 with EQQ was found to crosslink at half the rate of KCNQ1, which shows the association of the independent KCNE1 subunits into the unoccupied clefts in the EQQ channel complex. These findings differentiate the functionality of 2:4 KCNE1:KCNQ1 from a wild type channel complex and demonstrate that there is no intrinsic mechanism limiting the association of additional β-subunits up to four, confirming a variable stoichiometry model for IKs.