Individual IKs channels at the surface of mammalian cells contain two KCNE1 accessory subunits

Significance IKslow (IKs) channels allow heartbeats and hearing. The channels contain two types of subunits: Q1 and E1. Both are required. While four Q1 subunits are known to form the pore, the number of E1 subunits has been controversial for over a decade. The answer is critical to understanding cardiac function, diseases, including life-threatening arrhythmias, and for drug development. Here, we describe an improved method to study the composition and surface density of single IKs channels—simultaneous, two-color, subunit counting for channels on the surface of live mammalian cells. We show that IKs channels contain two E1 subunits and four Q1 subunits. This subunit ratio does not vary even when levels of E1 are raised or lowered 10-fold as found in tissues. KCNE1 (E1) β-subunits assemble with KCNQ1 (Q1) voltage-gated K+ channel α-subunits to form IKslow (IKs) channels in the heart and ear. The number of E1 subunits in IKs channels has been an issue of ongoing debate. Here, we use single-molecule spectroscopy to demonstrate that surface IKs channels with human subunits contain two E1 and four Q1 subunits. This stoichiometry does not vary. Thus, IKs channels in cells with elevated levels of E1 carry no more than two E1 subunits. Cells with low levels of E1 produce IKs channels with two E1 subunits and Q1 channels with no E1 subunits—channels with one E1 do not appear to form or are restricted from surface expression. The plethora of models of cardiac function, transgenic animals, and drug screens based on variable E1 stoichiometry do not reflect physiology.