Model for the evolution of Shaker-like Kv regulatory subunits adopting a 3:1R functional stoichiometry. (A) A precondition for the evolution of the regulatory phenotype in Shaker-like Kv family channels is coexpression of two subunits (A and B) from the same subfamily (Kv1, Kv2, Kv3, or Kv4) in at least a subset of cells. Each subunit forms homomeric channels, and because they have cross-compatibility, they can also form heterotetramers in four possible stoichiometries (3A:1B, 2A:2B adjacent, 2A:2B diagonal, and 1A:3B). All channels formed are functional, indicated by the presence of a K⁺ ion (black circle) in the pore. (B) Model 1, our favored model, provides a two-step path to evolution of the regulatory phenotype (in subunit B in this example) functioning in a 3:1R stoichiometry. In step 1, mutation(s) in subunit B establish self-incompatibility (shown here as mutations in T1, star), but do not eliminate cross-compatibility with subunit A. This restricts channel assembly to three possible stoichiometries (4A, 3A:1B, 2A:2B, diagonal); all assembled channels are functional. Expression of subunit B increases the number of channels formed (relative to expression of subunit A alone) and is therefore critical for maintaining currents at or near their premutation starting levels. Step 1 effectively established the regulatory phenotype for subunit B. In step 2 of the model, subunit B accumulates gate mutation(s) tolerated in only a single subunit (star), disrupting 2:2R assembly, conduction, or gating. This establishes 3:1R as the single functional heteromer stoichiometry. Loss of function in 2:2R channels is depicted by replacement of the K⁺ ion with a black X. Dominant-negative suppression of current will occur at highly subunit B–biased expression ratios as few A-A contacts will form during assembly, but reasonable currents will remain with balanced expression. (C) In the alternative model 2, subunit B gate incompatibility (star) evolves first, while self-compatibility remains. This results in strong dominant negative suppression because most subunits are tied up in nonfunctional channels. Model 2 is likely an evolutionary dead end because the gate mutation will engage negative selection to preserve current levels.