Nicotinic acetylcholine receptor of α7 type (α7-nAChR) presented in the nervous and immune systems and epithelium is a promising therapeutic target for cognitive disfunctions and cancer treatment. Weak toxin from Naja kaouthia venom (WTX) is a non-conventional three-finger neurotoxin, targeting α7-nAChR with weak affinity. There are no data on interaction mode of non-conventional neurotoxins with nAChRs. Using α-bungarotoxin (classical three-finger neurotoxin with high affinity to α7-nAChR), we showed applicability of cryo-EM to study complexes of α7-nAChR extracellular ligand-binding domain (α7-ECD) with toxins. Using cryo-EM structure of the α7-ECD/WTX complex, together with NMR data on membrane active site in the WTX molecule and mutagenesis data, we reconstruct the structure of α7-nAChR/WTX complex in the membrane environment. WTX interacts at the entrance to the orthosteric site located at the receptor intersubunit interface and simultaneously forms the contacts with the membrane surface. WTX interaction mode with α7-nAChR significantly differs from α-bungarotoxin’s one, which does not contact the membrane. Our study reveals the important role of the membrane for interaction of non-conventional neurotoxins with the nicotinic receptors.

Ly6/uPAR proteins regulate many essential functions in the nervous and immune systems and epithelium. Most of these proteins contain single β-structural LU domains with three protruding loops and are glycosylphosphatidylinositol (GPI)-anchored to a membrane. The GPI-anchor role is currently poorly studied. Here, we investigated the positional and orientational preferences of six GPI-anchored proteins in the receptor-unbound state by molecular dynamics simulations. Regardless of the linker length between the LU domain and GPI-anchor, the proteins interacted with the membrane by polypeptide parts and N-/O-glycans. Lynx1, Lynx2, Lypd6B, and Ly6H contacted the membrane by the loop regions responsible for interactions with nicotinic acetylcholine receptors, while Lypd6 and CD59 demonstrated unique orientations with accessible receptor-binding sites. Thus, GPI-anchoring does not guarantee an optimal ‘pre-orientation’ of the LU domain for the receptor interaction.

Cancer progression is characterized by microenvironmental acidification. Tumor cells adapt to low environmental pH by activating acid-sensing trimeric ion channels of the DEG/ENaC family. The α-ENaC/ASIC1a/γ-ENaC heterotrimeric channel is a tumor-specific acid-sensing channel, and its targeting can be considered a new strategy for cancer therapy. Mambalgin-2 from the Dendroaspis polylepis venom inhibits the α-ENaC/ASIC1a/γ-ENaC heterotrimer more effectively than the homotrimeric ASIC1a channel, initially proposed as the target of mambalgin-2. Although the molecular basis of such mambalgin selectivity remained unclear. Here, we built the models of the complexes of mambalgin-2 with the α-ENaC/ASIC1a/γ-ENaC and ASIC1a channels, performed MD and predicted the difference in the binding modes. The importance of the ‘head’ loop region of mambalgin-2 for the interaction with the hetero-, but not with the homotrimeric channel was confirmed by site-directed mutagenesis and electrophysiology. A new mode of allosteric regulation of the ENaC channels by linking the thumb domain of the ASIC1a subunit with the palm domain of the γ-ENaC subunit was proposed. The data obtained provide new insights into the regulation of various types of acid-sensing ion channels and the development of new strategies for cancer treatment.

TRPA1 is a homotetrameric non-selective calcium-permeable channel. It contributes to chemical and temperature sensitivity, acute pain sensation, and development of inflammation. HCIQ2c1 is a peptide from the sea anemone Heteractis magnifica that inhibits serine proteases. Here, we showed that HCIQ2c1 significantly reduces AITC- and capsaicin-induced pain and inflammation in mice. Electrophysiology recordings in Xenopus oocytes expressing rat TRPA1 channel revealed that HCIQ2c1 binds to open TRPA1 and prevents its transition to closed and inhibitor-insensitive ‘hyperactivated’ states. NMR study of the ¹⁵N-labeled recombinant HCIQ2c1 analog described a classical Kunitz-type structure and revealed two dynamic hot-spots (loops responsible for protease binding and regions near the N- and C-termini) that exhibit simultaneous mobility on two timescales (ps–ns and μs–ms). In modelled HCIQ2c1/TRPA1 complex, the peptide interacts simultaneously with one voltage-sensing-like domain and two pore domain fragments from different channel’s subunits, and with lipid molecules. The model explains stabilization of the channel in the open conformation and the restriction of ‘hyperactivation’, which are probably responsible for the observed analgetic activity. HCIQ2c1 is the third peptide ligand of TRPA1 from sea anemones and the first Kunitz-type ligand of this channel. HCIQ2c1 is a prototype of efficient analgesic and anti-inflammatory drugs.