Endogenous TRPV4 Expression of a Hybrid Neuronal Cell Line N18D3 and Its Utilization to Find a Novel Synthetic Ligand
Primary sensory afferent neurons detect environmental and painful stimuli at their peripheral termini. A group of transient receptor potential ion channels (TRPs) are expressed in these neurons and constitute sensor molecules for the stimuli such as thermal, mechanical, and chemical insults. We examined whether a mouse sensory neuronal line, N18D3, shows the sensory TRP expressions and their functionality. In Ca2+ imaging and electrophysiology with these cells, putative TRPV4-mediated responses were observed. TRPV4-specific sensory modalities including sensitivity to a specific agonist, hypotonicity, or an elevated temperature were reproduced in N18D3 cells. Electrophysiological and pharmacological profiles conformed to those from native TRPV4 of primarily cultured neurons. The TRPV4 expression in N18D3 was also confirmed by RT-PCR and Western blot analyses. Thus, N18D3 cells may represent TRPV4-expressing sensory neurons. Further, using this cell lines, we discovered a novel synthetic TRPV4-specific agonist, MLV-0901. These results suggest that N18D3 is a reliable cell line for functional and pharmacological TRPV4 assays. The chemical information from the novel agonist will contribute to TRPV4-targeting drug design.
KeywordsTRPV4 N18D3 Sensory neurons Novel ligand MLV-0901
This work was supported by grants from the National Research Foundation of Korea (NRF-2017R1A2B2001817, NRF-2017M3C7A1025600) and Korea Health Technology R&D Project of Ministry of Health & Welfare (HI15C2099).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
- Koh SH, Kim SH, Kwon H, Kim JG, Kim JH, Yang KH, Kim J, Kim SU, HJ Y, Do BR, Kim KS, Jung HK (2004) Phosphatidylinositol-3 kinase/Akt and GSK-3 mediated cytoprotective effect of epigallocatechin gallate on oxidative stress-injured neuronal-differentiated N18D3 cells. Neurotoxicology 25(5):793–802CrossRefPubMedGoogle Scholar
- Rimmerman N, Bradshaw HB, Hughes HV, Chen JS, SS H, McHugh D, Vefring E, Jahnsen JA, Thompson EL, Masuda K, Cravatt BF, Burstein S, Vasko MR, Prieto AL, O'Dell DK, Walker JM (2008) N-palmitoyl glycine, a novel endogenous lipid that acts as a modulator of calcium influx and nitric oxide production in sensory neurons. Mol Pharmacol 74(1):213–224CrossRefPubMedGoogle Scholar
- Ruan B, Pong K, Jow F, Bowlby M, Crozier RA, Liu D, Liang S, Chen Y, Mercado ML, Feng X, Bennett F, von Schack D, McDonald L, Zaleska MM, Wood A, Reinhart PH, Magolda RL, Skotnicki J, Pangalos MN, Koehn FE, Carter GT, Abou-Gharbia M, Graziani EI (2008) Binding of rapamycin analogs to calcium channels and FKBP52 contributes to their neuroprotective activities. Proc Natl Acad Sci U S A 105(1):33–38CrossRefPubMedGoogle Scholar
- Story GM, Peier AM, Reeve AJ, Eid SR, Mosbacher J, Hricik TR, Earley TJ, Hergarden AC, Andersson DA, Hwang SW, McIntyre P, Jegla T, Bevan S, Patapoutian A (2003) ANKTM1, a TRP-like channel expressed in nociceptive neurons, is activated by cold temperatures. Cell 112(6):819–829CrossRefPubMedGoogle Scholar