Molecular basis for a pore block of Tentonin 3 expressed in HEK293 cells by a conopeptide, NMB‐1

Background and Purpose Tentonin 3 (TTN3/TMEM150C) is a mechanosensitive ion channel that plays critical roles in mechanotransduction processes. TTN3 forms a tetramer with a predicted rectangular shape and a central pore. A conotoxin ρ‐TIA and its synthetic analog, noxious mechanosensation blocker 1 (NMB‐1), were initially developed to inhibit slowly adapting (SA) ‐type mechanically activated (MA) currents in dorsal root ganglion (DRG) neurons. Since TTN3 confers slowly‐adapting MA currents in DRG neurons, both NMB‐1 and ρ‐TIA were hypothesized to inhibit TTN3. Experimental Approach To record MA currents, a few micrometre step indentations were applied to HEK cells expressing TTN3. NMB‐1 and ρ‐TIA were applied to these cells to test their specific antagonism. Mutations of the conopeptides and TTN3 were made to identify underlying mechanisms of inhibition. Key Results NMB‐1 strongly inhibited TTN3, whereas ρ‐TIA had only a weak effect, and neither peptide affected Piezo channels. Alanine‐scanning mutagenesis coupled with electrophysiological assays pinpointed that a positively charged residue in NMB‐1 and ρ‐TIA is essential for their inhibitory action. Additionally, a glutamate residue (Glu126) near the pore entrance of TTN3 was identified as critical for the NMB‐1 inhibitory action, suggesting a key electrostatic interaction between NMB‐1 and TTN3. Molecular dynamics simulations further supported this electrostatic interaction between the peptide ligand and the channel protein. Conclusions and Implications NMB‐1 specifically blocks a mechanosensitive channel, TTN3, via electrostatic interaction. These findings offer mechanistic insights into the selective inhibition of TTN3 by NMB‐1 and provide a foundation for developing therapeutic agents targeting TTN3‐related channelopathies.