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.