Molecular assembly of the KCNQ1-KCNE1-BACE1 complex

Abstract

We previously showed that the {beta}-secretase BACE1 directly modulates KCNQ1 channels through a non-proteolytic mechanism. Here, we dissect the molecular interplay among KCNQ1, its canonical auxiliary subunit KCNE1, and BACE1 using bimolecular fluorescence complementation (BiFC), electrophysiology, single-molecule pull-down (SiMPull), and a Forster resonance energy transfer (FRET)-based interaction assay. BiFC validated KCNQ1 homotetramerization and confirmed specific interactions of KCNQ1 with KCNE1 and with BACE1 at the plasma membrane. To map interaction determinants, we generated six KCNE1/BACE1 chimeras. BiFC and electrophysiological recordings revealed domain-specific contributions: BACE1s large extracellular domain primarily mediates its modulatory effects on KCNQ1 gating, while--consistent with previous reports--the KCNE1 transmembrane segment is necessary and sufficient to confer an IKs-like phenotype, and the KCNE1 intracellular domain tunes the voltage dependence of activation. Notably, a chimera combining the BACE1 extracellular region with KCNE1 transmembrane and intracellular regions produced an IKs-like current with additional BACE1-like slowing of activation, indicating functional additivity. Stoichiometry measurements by SiMPull and bleaching-step analysis demonstrated that KCNQ1 channel complexes predominantly recruit two BACE1 molecules. Although BACE1 oligomerizes at the plasma membrane, BiFC and FRET showed that KCNQ1 co-expression reduces BACE1 homomeric assembly, and this effect is unchanged by KCNE1 co-expression. Together, our data support a model in which BACE1 binds directly to KCNQ1, occupies a site distinct from KCNE1, and modulates KCNQ1 gating via its extracellular domain while remaining compatible with KCNE1 co-assembly.