Inactivation mechanism of an immunoglobulin modulator of the T-cell potassium channel Kv1.3.

Abstract

Kv1.3 belongs to the Shaker family of voltage-activated potassium channels that are abundantly expressed in the T lymphocytes, where it plays an important role in T-cell activation. This role has made Kv1.3 a target for therapeutic immunomodulation for autoimmune diseases like multiple sclerosis, type 1 diabetes, and rheumatoid arthritis. Here, through cryo-electron microscopy (cryo-EM) and electrophysiology, we have investigated the modulatory mechanism of an inhibitory nanobody A0194009G09 (Ablynx/Sanofi). Functional studies reveal that the nanobody inhibits Kv1.3 by promoting slow C-type inactivation and that multiple nanobodies can bind to individual Kv1.3 channels. The structure of the human Kv1.3 in apo state (without modulators) showed a dynamic selectivity filter (SF) with two conformations (D1, D2) that are dilated when compared to reference structures where the SF is conducting. In the presence of the nanobody, the structure displays a SF in the D1 conformation, similar to the apo protein, but also a distinct conformation where the SF was further dilated (D3). Further, the nanobody-bound structure reveals how the nanobody bridges the turret loop within the pore domain and the S1-S2 loop within the voltage sensing domains to promote C-type inactivation. To explore how the nanobody promotes inactivation, we mutated residues at the interface between the nanobody and channel in both the S1-S2 loop and the turret loop, as well as a residue in the SF (H451) known to control inactivation in Kv1.3. Interestingly, the mutations that prevent inactivation in Kv1.3 largely attenuated the inhibitory action of the nanobody, similar to the mutations at the Kv1.3-nanobody binding interface. Taken together, our results demonstrate that the nanobody inhibits Kv1.3 by promoting inactivation, and we are currently solving mutant structures to elucidate the molecular mechanisms of how nanobody binding promotes inactivation.