Structural basis of connexin-36 gap junction channel inhibition

Abstract

Connexin gap junction channels and hemichannels play important roles in intercellular communication and signaling. Some of connexin isoforms are associated with diseases, including hereditary neuropathies, heart disease and cancer. Although small molecule inhibitors of connexins show promise as therapeutic agents, the molecular mechanisms of connexin channel inhibition are unknown. Here, we report the cryo-EM structure of connexin-36 (Cx36) bound to an anti-malarial drug mefloquine at 2.1 Å resolution. Six drug binding sites partially occlude the pore of each connexon forming the channel. Each drug molecule in the ring makes contacts with residues in the pore-lining pocket and with the neighbouring mefloquine molecules, partially occluding the pore and modifying the pore electrostatics, ultimately reducing solute translocation through the channel. Structures of Cx36 in the presence of quinine and quinidine show a similar mode of drug binding. Molecular dynamics simulations of Cx36 bound to mefloquine show that drug binding affects the kinetics of ion passage through the pore. This previously undescribed mode of connexin channel inhibition presents an opportunity for designing subtype-specific connexin inhibitors.One-sentence summaryMechanism of connexin channel inhibition by small molecules

Mefloquine-induced conformational shift in Cx36 N-terminal helix leading to channel closure mediated by lipid bilayer

“…1a, b, upper boxes). While investigating the inhibition mechanism of Cx36 by MFQ and AA, the Cx36-MFQ complex and the Cx32-MFQ complex in detergent micelles were recently reported, showing only the FN state, both in the presence and absence of MFQ 31,32 . This observation aligns with our previously determined structures of human Cx36 in detergents 17 , suggesting that detergents significantly influence the conformation equilibrium between the PLN and FN states.…”