Lim, Sujin Woo, Junhyuk Pak, Sungmin Paik, Joonho Hong, Gyu-Sang Wi, Ji Hun Lee, Sanghee Fenollar-Ferrer, Cristina Han, Kyungreem Shin, Young Kee Oh, Uhtaek 2025-12-03T06:00:13Z 2025-12-03T06:00:13Z 2025-12-03 2025-11 0007-1188 https://pubs.kist.re.kr/handle/201004/153740 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. English Wiley-Blackwell Molecular basis for a pore block of Tentonin 3 expressed in HEK293 cells by a conopeptide, NMB-1 Article 10.1111/bph.70278 1 British Journal of Pharmacology British Journal of Pharmacology N scie scopus Pharmacology & Pharmacy Pharmacology & Pharmacy Article; Early Access MECHANOSENSITIVE ION CHANNELS PEPTIDE FORCE VANILLOID RECEPTOR POLYMERIZATION JASPLAKINOLIDE CONCISE GUIDE IDENTIFICATION SOFTWARE PIEZO2 conotoxin electrostatic interaction mechanosensitive channel molecular dynamics simulation NMB-1 Tentonin 3 TMEM150C