Rearrangement of a unique Kv1.3 selectivity filter conformation upon binding of a drug

We report two structures of the human voltage-gated potassium channel (Kv) Kv1.3 in immune cells alone (apo-Kv1.3) and bound to an immunomodulatory drug called dalazatide (dalazatide–Kv1.3). Both the apo-Kv1.3 and dalazatide–Kv1.3 structures are in an activated state based on their depolarized voltage sensor and open inner gate. In apo-Kv1.3, the aromatic residue in the signature sequence (Y447) adopts a position that diverges 11 Å from other K⁺ channels. The outer pore is significantly rearranged, causing widening of the selectivity filter and perturbation of ion binding within the filter. This conformation is stabilized by a network of intrasubunit hydrogen bonds. In dalazatide–Kv1.3, binding of dalazatide to the channel’s outer vestibule narrows the selectivity filter, Y447 occupies a position seen in other K⁺ channels, and this conformation is stabilized by a network of intersubunit hydrogen bonds. These remarkable rearrangements in the selectivity filter underlie Kv1.3’s transition into the drug-blocked state.

Potassium channels form K⁺-selective pores that span cell membranes in virtually all living organisms. In humans, a family of 78 genes encodes four classes of K⁺ channels (voltage-gated, calcium-activated, inward rectifier, and two-pore channels), which are involved in a multitude of physiological functions in both electrically excitable and nonexcitable cells (1). All four classes of channels conduct K⁺ ions selectively and rapidly, but they differ in how they are gated. The selectivity filter is the structural element responsible for the exquisitely K⁺-selective pore (2–5). It is the narrowest part of the ion conduction pathway and connects a water-filled cavity in the center of the protein with an outer vestibule in the extracellular solution. The filter accommodates K⁺ ions at four sites called S1, S2, S3, and S4 starting at the extracellular side. The signature sequence G(Y/F)G in the selectivity filter plays a critical role in making the pore K⁺ selective (6, 7). In all K⁺ channel structures determined, both bacterial and eukaryotic, the aromatic residue (Y or F) in the signature sequence is nearly identical in position, although these channels differ in the conformation (closed or open) of the S6 helical inner gate (8). In the hERG/Kv11.1 channel, a subtle deviation in the position of F627 in the signature sequence causes a slight widening of the selectivity filter, which has been suggested to underlie the channel’s transition into the C-type inactivated state (8).The voltage-gated potassium channel (Kv) Kv1.3–Kvβ2 in lymphocytes and microglia provides the counterbalancing cation efflux to promote calcium entry necessary for calcium signaling (9, 10). Selective blockers of Kv1.3–Kvβ2 treat diverse autoimmune and neuroinflammatory diseases in rodent models (9, 10), highlighting the channel’s physiological and pharmacological importance. Here, we determined structures of Kv1.3 complexed to its accessory subunit Kvβ2 alone (apo-Kv1.3) and bound to dalazatide (dalazatide–Kv1.3), a potent and selective peptide inhibitor of Kv1.3 in clinical trials for autoimmune and neuroinflammatory diseases (10–14). Both apo-Kv1.3 and dalazatide–Kv1.3 are in the activated state based on the depolarized voltage sensor and open S6 helical inner gate. Comparison of the two structures reveals substantial conformational changes in the selectivity filter. In apo-Kv1.3, Y447 in the signature sequence diverges more than 11 Å from the position of corresponding aromatic residues in other K⁺ channels, both in eukaryotes and bacteria. The outer pore is wider at S1 and S2 and narrowed at S0 K⁺-binding sites, resulting in loss of the K⁺ ion from site S2. A network of intrasubunit hydrogen bonds (H451–Y447, H451–D449) stabilizes this unique conformation of the selectivity filter of apo-Kv1.3, and, interestingly, the intrasubunit hydrogen bond (W436–D449) that prevents C-type inactivation (15) is absent. Apo-Kv1.3’s selectivity filter and voltage-sensing domain (VSD) differ significantly from two structures of Kv1.3 that were recently described (16). In dalazatide–Kv1.3, dalazatide’s interaction with H451 disrupts the H451–Y447 hydrogen bond, freeing Y447 to swing back into the interior of the selectivity filter and adopt a position seen in other K⁺ channels. The selectivity filter is narrower, and K⁺ ions are present at sites S2–S4 but not at S1. This conformation is stabilized by a network of intersubunit hydrogen bonds (Y447–W437, Y447–T441, and H451–D449), but the intrasubunit hydrogen bond (W436–D449) that prevents C-type inactivation (15) is likely absent. Our structures provide a basis for the design of Kv1.3 inhibitors for use as immunomodulatory therapeutics.