a, Current traces for the structural L403A construct of Kv2.1 (1–598) recorded in 2 mM external K+ from −100 mV to +60 mV (20 mV increments) using a holding voltage of −100 mV. Red dotted line denotes zero current. b, Conductance–voltage (G–V) relations and voltage–steady-state inactivation relations (I–V) obtained from a three-pulse protocol (Fig. 3d) comparing the L403A Kv2.1 (1–598) (G–V, V1/2 = 56.7 ± 1.2 mV, z = 1.5 ± 0.1, n = 5 cells in two independent experiments; I–V (inactivation), V1/2 = −6.9 ± 0.7, z = 4.6 ± 0.1, n = 7 cells in two independent experiments) with the L403A mutant in the full-length Kv2.1 channel (Fig. 3c,f). G–V relations were obtained from tail currents using a holding voltage of −90 mV, 20 ms voltage steps to between −50 mV and +120 mV (10 mV increments) and a tail voltage of 0 mV. Solid symbols represent mean and solid lines corresponds to fits of the Boltzmann equation. Error bars are s.e.m. c, Side (left) and external (right) views of the Kv2.1-L403A EM map, with each subunit shown in different colours. EM densities that could correspond to lipids are in yellow. d, Model of the ion selectivity filter superimposed with the EM map, with the K+ ion densities highlighted in green. e, Superimposition of the most inactivated subunit of the L403A mutant (protomer D) with one subunit of Kv2.1 illustrating conformational changes in S6, S5 and the S4–S5 linker. f, Conformational changes in the hydrophobic coupling nexus between Kv2.1 and the L403A mutant protomer D. g, Superimposition of Kv2.1 and the L403 mutant structures viewed from the intracellular side of the membrane with key residues in S6 shown in stick representation. h, Superimposed views of the S6 helices of Kv2.1 and the most inactivated subunit (protomer D) of the L403A mutant. i, HOLE representations of the ion permeation pathway for Kv2.1 and the L403A mutant with the backbone for S6 and the selectivity filter of the models shown for reference. j, Plot of pore radius along the ion permeation pathway with dashed green line at the radius of a hydrated K+ ion.