Charybdotoxin Binding in the I Ks Pore Demonstrates Two MinK Subunits in Each Channel Complex

Abstract

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, 3H-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).