Predicted stoichiometry and abundance of surface-expressed channels for Kv2.1:Kv6.4 and Kv6.4-PIPIIV-Kv2.1CT at various expression ratios. Surface-expressed channel tetramers are depicted as open circles and colored-in quarters according to subunit composition (Kv2.1, black; Kv6.4, magenta; Kv6.4-PIPIIV-Kv2.1CT, blue). Monomer abundance is shown below the channels; Kv2.1 abundance remains constant while Kv6.4 abundance is increased from left to right. Relative abundance for each stoichiometry of surface-expressed channel and each type of monomer is represented by the size of the respective icon. (A) For Kv2.1:Kv6.4, the number of 3:1R heteromers initially increases as Kv6.4 expression is increased, but then drops as availability of Kv2.1 homodimers, which are required for 3:1R formation, becomes limiting at the highest Kv6.4 expression levels. TIRF results show that 2:2R formation is inefficient and never exceeds 30% of the total heteromer population, even at the highest Kv6.4 expression levels. Therefore, the total number of surface-expressed channels will reduce and become very small as Kv6.4 expression is increased against a constant level of Kv2.1 expression. (B) For Kv2.1:Kv6.4-PIPIIV-Kv2.1CT, channel abundance is predicted to decrease less precipitously as Kv6.4-PIPIIV-Kv2.1CT expression level increases because 2:2R formation is more efficient, reaching ∼60% of all heteromers at the most Kv6.4-PIPIIV-Kv2.1CT–biased expression ratios. Assuming similar levels of 3:1R formation, this represents an approximately fourfold increase in the number of 2:2R heteromers formed compared with Kv6.4.