Charybdotoxin binding in the IKs pore demonstrates two MinK subunits in each channel complex

I-Ks voltage-gated K+ channels contain four pore-forming KCNQ1 subunits and MinK accessory subunits in a number that has been controversial. Here, I-Ks channels assembled naturally by monomer subunits are compared to those with linked subunits that force defined stoichiometries. Two strategies that exploit charybdotoxin (CTX)-sensitive subunit variants are applied. First, CTX on rate, off rate, and equilibrium affinity are found to be the same for channels of monomers and those with a fixed 2:4 MinK:KCNQ1 valence. Second, H-3-CTX and an antibody are used to directly quantify channels and MinK subunits, respectively, showing 1.97 +/- 0.07 MinK per I-Ks channel. Additional MinK subunits do not enter channels of monomeric subunits or those with fixed 2:4 valence. We conclude that two MinK subunits are necessary, sufficient, and the norm in I-Ks channels. This stoichiometry is expected for other K+ channels that contain MinK or MinK-related peptides (MiRPs).