Structural homology models of Kv2.1 homomers and Kv2.1:6.4 heteromers suggest the Kv6.4 activation gate might perturb pore stability and function. (A) Sequence alignment of the Kv1.2, Kv2.1, and Kv6.4 PDs. Transmembrane domains S5 and S6 and the K⁺ selectivity filter are underlined. Residues identical or conservatively substituted across all three sequences are shaded magenta and black, respectively. The six–amino acid activation gate is boxed with positions 1–6 labeled. Note there are identical signpost residues throughout the PDs allowing precise alignment between Kv1.2 (determined structure, Long et al., 2005a), Kv2.1, and Kv6.4 (structural models based on Kv1.2 presented here). (B–E) Snapshots of the closed conduction pathway viewed from the extracellular side for structural models of a Kv2.1 homomer (B), a Kv2.1:Kv6.4 3:1R heteromer (C), a Kv2.1:Kv6.4 2:2R heteromer (D), and a Kv2.1:Kv6.4 2:2R heteromer with the Kv2.1 activation gate (PIPIIV) substituted for the Kv6.4 activation gate (PATSIF; E). The protein backbone (thin tubes) is colored white for Kv2.1 and light purple for Kv6.4. Side chains (van der Waals representation) are shown for positions 2, 3, and 6 of the activation gate and colored according to hydrophobicity index in the Kyte and Doolittle scale with their values ranging from 1.8 for alanine (green) to 4.5 for isoleucine (white; scale provided below panels). Residues that line the conduction pathway (I2 and V6 in Kv2.1; A2 and F6 in Kv6.4) are labeled in C. Yellow ribbons in all panels highlight the position of the gate backbone. In Kv2.1, there is an expected intersubunit hydrophobic vapor lock at the activation gate. While insertion of a single Kv6.4 subunit (C) is well tolerated, insertion of two diagonally opposed Kv6.4 subunits (D) simultaneously introduces a hydrophilic cleft that bisects the gate and increases the distance between diagonally opposed subunits. Both changes favor disruption of the intersubunit vapor lock and could hypothetically reduce tetramer stability at the gate intersubunit interface. (F–M) The bottom two rows are the view from the cytoplasmic side—one row for the closed state (F–I) and another for the open (J–M). Side chains at the gate constriction are shown in space fill and colored by the residue type (Kv2.1: I2, white; P3, cyan; V6 gray; Kv6.4: A2, gray; T3, brown; F6, pink). In the closed models, the hydroxyl group of T3 in the Kv6.4 gate faces the neighboring subunit (G and H) and thus contributes to the hydrophilic cleft observed in the extracellular view of the 2:2R tetramer (D). In the open conformations, a dehydrated K⁺ ion (CHARMM36 radius, 1.76 Å) is depicted with a blue circle in the pore opening at the narrowest point of the conduction pathway in the activation gate region. A magenta dashed line surrounds the portion of the opening accessible to the center of the dehydrated K⁺ ion (magenta dot) as determined by rolling the ion against the side chains lining the pore. Blue mesh covers the inaccessible region of the pore. The black circle around the ion roughly approximates the radius of the first hydration shell (i.e., K⁺ radius plus the diameter of the TIP3P water molecule in CHARMM36). While not an explicit simulation in an all-atom setting, it is nevertheless obvious that F6 in the Kv6.4 activation gate narrows the gate opening proportional to the number of Kv6.4 gates. In the 2:2R conformation, almost complete dehydration of a K⁺ ion would be needed for passage. Alternatively, more drastic rearrangements of the backbone than can be observed in our short vacuum simulations might reduce the constriction, but still block conduction by disrupting gating as observed for the F6 substitution in Shaker (Kitaguchi et al., 2004). Note that substitution of the Kv2.1 activation gate into Kv6.4 in the 2:2R simulation restores both the hydrophobic vapor lock of the closed state (E and I) and a wide conduction pathway to the open state (M).