Primary cell-based high-throughput screening assay to identify agonists of the transient receptor potential channel N1 (TRPN1)
Name: Primary cell-based high-throughput screening assay to identify agonists of the transient receptor potential channel N1 (TRPN1) ..more
BioActive Compounds: 390
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: Stefan Heller, Stanford University
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number 1 R03 MH083077-01
Grant Proposal PI: Stefan Heller, Stanford University
External Assay ID: TRPN1_AG_Calcium_1536_%ACT
Name: Primary cell-based high-throughput screening assay to identify agonists of the transient receptor potential channel N1 (TRPN1)
Cell signaling pathways that mediate osmosensation, photosensation, and thermosensation depend on a family of diverse transient receptor potential (TRP) cation channels, which are activated by agonist-receptor coupling (1-4). A role for these channels in inner ear hair cell mechanotransduction was gleaned from TRP channel mutations identified in flies, worms, and lower vertebrates with defective balance and impaired sensitivity to touch (1-4). Studies suggest that TRPML3 and TRPN1 channels have an important role in hearing and balance (4-7). Although TRPN1 is not present in the genomes of higher eukaryotes (8), TRPN1 orthologs were discovered in zebrafish (Danio rerio) and frog (Xenopus laevis) (9, 10). Studies showing that TRPN1-deficient zebrafish exhibit defective hair cell electrical traces, deafness and atypical swimming behavior resulting from impaired balance, suggest that TRPN1 has auditory and vestibular functions (10). Thus, the identification of selective probes for TRPN1 would be useful to investigate the process of inner ear mechanotransduction.
1. Clapham, D.E., TRP channels as cellular sensors. Nature. 2003. 426(6966): p. 517-24.
2. Cuajungco, M.P., C. Grimm, and S. Heller, TRP channels as candidates for hearing and balance abnormalities in vertebrates. Biochim Biophys Acta. 2007. 1772(8): p. 1022-7.
3. Gillespie, P.G. and R.G. Walker. Molecular basis of mechanosensory transduction. Nature. 2001. 413(6852): p. 194-202.
4. Eberl, D.F., R.W. Hardy, and M.J. Kernan. Genetically similar transduction mechanisms for touch and hearing in Drosophila. J Neurosci. 2000. 20(16): p. 5981-8.
5. Gong, Z., W. Son, Y.D. Chung, J. Kim, D.W. Shin, C.A. McClung, Y. Lee, H.W. Lee, D.J. Chang, B.K. Kaang, H. Cho, U. Oh, J. Hirsh, M.J. Kernan, and C. Kim. Two interdependent TRPV channel subunits, inactive and Nanchung, mediate hearing in Drosophila. J Neurosci. 2004. 24(41): p. 9059-66.
6. Kim, J., Y.D. Chung, D.Y. Park, S. Choi, D.W. Shin, H. Soh, H.W. Lee, W. Son, J. Yim, C.S. Park, M.J. Kernan, and C. Kim. A TRPV family ion channel required for hearing in Drosophila. Nature. 2003. 424(6944): p. 81-4.
7. Walker, R.G., A.T. Willingham, and C.S. Zuker. A Drosophila mechanosensory transduction channel. Science. 2000. 287(5461): p. 2229-34.
8. Corey, D.P. What is the hair cell transduction channel? J Physiol. 2006. 576(Pt 1): p. 23-8.
9. Shin, J.B., D. Adams, M. Paukert, M. Siba, S. Sidi, M. Levin, P.G. Gillespie, and S. Grunder. Xenopus TRPN1 (NOMPC) localizes to microtubule-based cilia in epithelial cells, including inner-ear hair cells. Proc Natl Acad Sci U S A. 2005. 102(35): p. 12572-7.
10. Sidi, S., R.W. Friedrich, and T. Nicolson. NompC TRP channel required for vertebrate sensory hair cell mechanotransduction. Science. 2003. 301(5629): p. 96-99.
TRPN1, TRP cation channel subfamily N, member 1, nompC, zebrafish, HEK 293, HTS assay, 1536, primary, agonist, activator, deafness, fluorescence, calcium, Fluo-8 dye, Scripps, Scripps Florida, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this assay is to identify test compounds that act as agonists of the TRPN1 cation channel. This assay employs a HEK293 cell line that stably expresses the zebrafish TRPN1-YFP cation channel. The cells are treated with test compounds followed by measurement of intracellular calcium as monitored by a fluorescent, cell permeable calcium indicator dye. As designed, compounds that act as TRPN1 agonists will increase calcium mobilization, resulting in increased relative fluorescence of the indicator dye, and thus increase well fluorescence.
The TRPN1 HEK293 cell line was routinely cultured in T-175 sq cm flasks at 37 degrees C and 95% relative humidity (RH). The growth media consisted of Minimum Essential Medium with GlutaMAX and supplemented with 10% v/v heat-inactivated qualified fetal bovine serum, 800 micrograms/mL Geneticin, and 1X antibiotic mix (penicillin, streptomycin, and neomycin). The day before the assay 1500 cells in 3 ul of growth media were seeded into each well of 1536 well microtiter plates and allowed to incubate at 37 degrees C, 5% CO2, and 95 % RH for 23 hours. Next, 2 ul of the fluorogenic Fluo-8 intracellular calcium indicator mixture with 1 mM trypan red plus (prepared according to the manufacturer's protocol) was added to each well. After a 1 hour incubation at 37 degrees C, 5% CO2, and 95 % RH followed by a 30 minute incubation at room temperature, the assay was started by performing a basal read of plate fluorescence (470-495 nm excitation and 515-575 nm emission) for 5 seconds on the FLIPR Tetra (Molecular Devices). Next, 15 nL of test compound (3 uM final nominal concentration) in DMSO, DMSO alone (0.3% final concentration), or the cholinergic agonist carbachol (87 uM final concentration) in DMSO were dispensed to the appropriate wells. Then a real time fluorescence measurement was immediately performed for the remaining 120 seconds of the assay.
A ratio for each well was calculated to normalize assay data, according to the following mathematical expression:
Ratio = I_Max / I_Min
Where I_Max represents the maximum measured fluorescence emission intensity over the 125 second read and I_Min represents the minimum (basal) measured fluorescence emission intensity before compound was added.
Percent activation was calculated from the median ratio as follows:
%Activation = ((Ratio_Test_Compound- Median_Ratio_Low_Control) / (Median_Ratio_High_Control - Median_Ratio_ Low_Control)) *100
Test_Compound is defined as wells containing test compound.
High_Control is defined as wells containing carbachol.
Low_Control is defined as wells containing DMSO.
A mathematical algorithm was used to determine nominally activating compounds in the primary screen. Two values were calculated for each assay plate: (1) the average percent activation of test compound wells and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter for each plate, i.e. any compound that exhibited greater % activation than that particular plate's cutoff parameter was declared active.
The reported Pubchem Activity Score has been normalized to 100% of the highest observed primary activation value. Negative % activation values are reported as activity score zero.
The inactive compounds of this assay have activity score range of 0 to 1 and active compounds range of activity score is 1 to 100.
List of Reagents:
TRPN1 HEK293 cell line (provided by Prof. Stefan Heller)
Fluo-8 No Wash Calcium Assay Kit (ABD Bioquest, part 36316)
Trypan red plus (ABD Bioquest, part 2456)
MEM with GlutaMAX (Invitrogen, part 41090-101)
Geneticin (Invitrogen, part 10131-027)
Trypsin-EDTA solution (Invitrogen, part 25200-056)
Fetal Bovine Serum (Invitrogen, part 16140-071)
Carbachol (Sigma, part C4382)
100X Penicillin-Streptomycin-Neomycin mix (Invitrogen, part 15640-055)
T-175 tissue culture flasks (Corning, part 431080)
1536-well plates (Greiner, part 789072)
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each separate campaign were assigned "Active/Inactive" status based upon that campaign's specific compound activity cutoff value. In this assay carbachol had an approximate EC50 of 1.5 uM. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that non-specifically modulate calcium levels or channel activity, and compounds that quench or emit fluorescence within the well. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary based upon the actual sample provided by the MLSMR.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)