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BioAssay: AID 2719

Late stage results from the probe development effort to identify selective agonists of the Transient Receptor Potential Channels 3 (TRPML3): Fura-2 profiling assay

Name: Late stage results from the probe development effort to identify selective agonists of the Transient Receptor Potential Channels 3 (TRPML3): Fura-2 profiling assay. ..more
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 Tested Compounds
 Tested Compounds
All(3)
 
 
Active(3)
 
 
 Tested Substances
 Tested Substances
All(3)
 
 
Active(3)
 
 
AID: 2719
Data Source: The Scripps Research Institute Molecular Screening Center (TRPML3_AG_FURA-2 LATE STAGE)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-03-30

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 3
Related Experiments
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AIDNameTypeProbeComment
1424Primary cell-based high-throughput screening assay to identify agonists of the transient receptor potential channel N1 (TRPN1)Screening depositor-specified cross reference: Primary Assay (TRPML3 agonists, 1X%ACT)
1448Primary cell-based high-throughput screening assay to identify agonists of the transient receptor potential channel ML3 (TRPML3)Screening depositor-specified cross reference: Counterscreen Assay (TRPN1 agonists, 1X%ACT)
1525Counterscreen assay for TRPML3 agonists: cell-based high-throughput screening assay to identify agonists of the transient receptor potential channel N1 (TRPN1)Screening depositor-specified cross reference: Counterscreen Assay (TRPN1 agonists, 3X%ACT)
1526Confirmation cell-based high-throughput screening assay for agonists of the transient receptor potential channel ML3 (TRPML3)Screening depositor-specified cross reference: Confirmation Assay (TRPML3 agonists, 3X %ACT)
1562Dose response cell-based high-throughput screening assay for agonists of the transient receptor potential channel ML3 (TRPML3)Confirmatory depositor-specified cross reference: Dose Response (TRPML3 agonists, 3X EC50)
1682Fluorescence counterscreen assay for TRPML3 agonists: dose response cell-based high-throughput screening assay to identify agonists of the transient receptor potential channel N1 (TRPN1)Confirmatory depositor-specified cross reference: Dose Response Counterscreen (TRPN1 agonists, 3X EC50)
1809Summary of probe development efforts to identify agonists of the transient receptor potential channel ML3 (TRPML3)Summary2 depositor-specified cross reference: Summary (TRPML3 agonists)
2510Late stage results from the probe development effort to identify selective agonists of the Transient Receptor Potential Channels 3 (TRPML3): fluorescence-based cell-based dose response assay for TRPML3 agonistsConfirmatory depositor-specified cross reference: Dose Response (TRPML3 agonists, 3X EC50)
2583Late stage counterscreen for the probe development effort to identify selective agonists of the Transient Receptor Potential Channels 3 (TRPML3): fluorescence-based cell-based dose response assay for TRPN1 agonists.Confirmatory depositor-specified cross reference: Dose Response Counterscreen (TRPN1 agonists, 3X EC50)
2692Late stage counterscreen results from the probe development effort to identify selective agonists of the Transient Receptor Potential Channels 3 (TRPML3): TRPN1 patch clamp assayScreening depositor-specified cross reference: TRPN1 Patch clamp assay
2116Late stage results from the probe development efforts to identify agonists of the Transient Receptor Potential Channels 3 and 2 (TRPML3 and TRPML2).Screening same project related to Summary assay
2694Late stage results from the probe development effort to identify selective agonists of the Transient Receptor Potential Channels 3 (TRPML3): TRPML3 patch clamp assayScreening same project related to Summary assay
2770Late stage counterscreen results from the probe development effort to identify selective agonists of the Transient Receptor Potential Channels 3 (TRPML3): other ion channel Fura-2 profiling assayScreening same project related to Summary assay
602128Late stage results from the probe development efforts to identify selective agonists of the Transient Receptor Potential Channels 3 (TRPML3): fluorescence-based cell-based dose response assay for TRPML3 agonists (probe candidates and analogs, Round 2)Confirmatory same project related to Summary assay
602129Late stage results from the probe development efforts to identify selective agonists of the Transient Receptor Potential Channels 3 (TRPML3): fluorescence-based cell-based dose response assay for TRPML3 agonists (probe candidates and analogs, Round 3)Confirmatory same project related to Summary assay
Description:
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: TRPML3_AG_FURA-2 LATE STAGE

Name: Late stage results from the probe development effort to identify selective agonists of the Transient Receptor Potential Channels 3 (TRPML3): Fura-2 profiling assay.

Description:

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 (1-5). 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 (1-5). TRPML3 (mucolipin 3; MCOLN3) is a TRP channel expressed in inner ear hair cells and stereocilia (5-7), suggesting it may play a role in hearing and mechanotransduction. Reports that mice with mutations in TRPML3 (known as varitint-waddler mutants) exhibit early-onset hearing loss accompanied by head-bobbing and circling behaviors (8-10), provide further support for a role of TRPML3 in hearing and vestibular function. As a result, the identification of selective probes for TRPML3 would be useful to investigate the function of TRPML3 in inner ear mechanotransduction and hearing biology (11).

References:

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. 96-9.
11. Small molecule activators of TRPML3. Grimm C, Jors S, Saldanha SA, Obukhov AG, Pan B, Oshima K, Cuajungco MP, Chase P, Hodder P, Heller S. Chem Biol. 2010 Feb 26;17(2):135-148.

Keywords:

Late stage, probes, selective, TRPML3, TRP, cation channel, ion channel, mucolipin 3, MCOLN3, deafness, HEK 293, agonist, agonism, activator, activation, activate, FURA-2, calcium, dye, intracellular, profiling, fluorescence, cells, cell-based, counterscreen, assay provider, assay, Scripps, Scripps Florida, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
Protocol
The purpose of these assays is to determine whether compounds identified as probe candidates are able to increase whole cell Ca2+ influx in HEK293 cells transfected with human TRPML3, other human, or murine (m) TRP channels, or zebrafish TRPN1. In this assay cells transiently expressing channels or YFP control plasmid are perfused with test compound, followed by measurement of intracellular [Ca2+] for 2 minutes with the fluorescent indicator fura-2-AM. Fura-2-AM is the membrane-permeable derivative of Fura-2. Compounds are added to cells 20-25 hours after transfection. Values are reported as mean values +/- SEM (n ≥ 3 independent experiments with 20-30 cells). The % activation values for TRPML2 in the SAR tables were calculated by normalizing the TRPML2 response ratios to TRPML3 response ratios. Compounds are tested at 10 micromolar. Please see reference 11 for details.
Protocol Summary:
This assay measures increases of intracellular calcium in TRPML3-transfected cells using fura-2 as the calcium indicator dye. Measurements of [Ca2+]i with the fluorescent indicators fura-2-AM was performed using a monochromator-based imaging system (iMIC platform and Polychrome V monochromator, TILL Photonics). HEK293 cells, plated onto glass coverslips, were loaded with 4 micromolar fura-2-AM in a standard bath solution (SBS) containing 138 mM NaCl, 6 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM HEPES, and 5.5 mM d-glucose (adjusted to pH 7.4 with NaOH) (11).
PubChem Activity Outcome and Score:
In this assay the PubChem Activity Score is assigned a value of 100 for probe compounds, 50 for actives and 0 for inactives. There are no inactive compounds.
List of Reagents:
TRPML3 HEK293 cell line (provided by Prof. Stefan Heller)
Fura-2-AM (Invitrogen)
Comment
Probes were identified. This assay was performed by the assay provider.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Cell Type: HEK293
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1deltaF (Average) [340 nm/380 nM] (10μM**)The ratio of emissions at 340nm and 380 nm wavelengths, in response to 10 micromolar test compound. Average of >= 3 measurements.Float
2deltaF (StdErr) [340 nm/380 nM]Standard Error of the ΔF measurement.Float
3Fura-2 Profiling AssignmentString
4Probe Molecule OutcomeIndicates whether the compound is assigned to be a probe or not. One of "Probe", "Analog", "Active" or "Inactive".String

** Test Concentration.
Additional Information
Grant Number: 1 R03 MH083077-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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