Khanra, N. | Search bioRxiv

bioRxiv2025

Structure of the human TWIK-2 potassium channel and its inhibition by pimozide

The potassium channel TWIK-2 is crucial for ATP-induced activation of the NLRP3 inflammasome in macrophages. The channel is a member of the two-pore domain potassium (K2P) channel superfamily and an emerging therapeutic target to mitigate severe inflammatory injury involving NLRP3 activation. We report the cryo-EM structure of human TWIK-2. In comparison to other K2P channels, the structure reveals a unique up conformation of Tyr111 in the selectivity filter and a SF1-P1 pocket behind the filter that could serve as a binding site for channel modulators. Density for acyl chains is present in fenestrations within the transmembrane region that connect the central cavity of the pore to the lipid membrane. Limited pharmacological tools are available for TWIK-2 despite its importance as a drug target. We show that the small molecule pimozide inhibits TWIK-2 and determine a structure of the channel with pimozide. Pimozide displaces the acyl chains and binds below the selectivity filter to block ion conduction. The drug may access its binding site via the membrane, suggesting that other hydrophobic small molecules could have utility for inhibiting TWIK-2. The work defines the structure of TWIK-2 and provides a structural foundation for development of specific inhibitors with potential utility as anti-inflammatory drugs. Significance StatementThe TWIK-2 potassium channel is a member of the two-pore domain potassium (K2P) channel superfamily and a potential therapeutic target to control severe inflammatory injury involving the NLRP3 inflammasome. We report the cryo-EM structure of the human TWIK-2 channel at 2.85 [A] resolution, revealing differences in comparison to other K2P channels. We identify that pimozide, an FDA-approved drug for Tourette syndrome, inhibits TWIK-2. A cryo-EM structure of TWIK-2 in complex with pimozide identifies its binding location and mechanism of inhibition. The work provides a structural foundation for development of specific TWIK-2 inhibitors that have potential therapeutic utility for inflammatory diseases involving NLRP3 activation.

biochemistry↗

bioRxiv2022

Structures of the T cell potassium channel Kv1.3 with immunoglobulin modulators

The Kv1.3 potassium channel is expressed abundantly on activated T cells and mediates the cellular immune response. This role has made the channel a target for therapeutic immunomodulation to block its activity and suppress T cell activation. We determined structures of human Kv1.3 alone, with a nanobody inhibitor, and with an antibody-toxin fusion blocker. Rather than block the channel directly, four copies of the nanobody bind the tetramers voltage sensing domains and the pore domain to induce an inactive pore conformation. In contrast, the antibody-toxin fusion docks its toxin domain at the extracellular mouth of the channel to insert a critical lysine into the pore. The lysine induces an active conformation of the pore yet blocks ion permeation. This study visualizes Kv1.3 pore dynamics, defines two distinct mechanisms to suppress Kv1.3 channel activity with exogenous inhibitors, and provides a framework to aid development of emerging T cell immunotherapies.

biophysics↗