Late 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 assay
Name: Late 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 assay. ..more
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: TRP_AG_FURA-2 SAR_Round_1
Name: Late 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 assay.
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).
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.
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The purpose of these assays is to determine whether compounds identified as selective TRPML3 agonist probe candidates are nonselective due to increasing whole cell Ca2+ influx in HEK293 cells that were either mock transfected or transfected with other human, murine, or zebrafish TRP channels. In this assay cells transiently expressing channels 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). Compounds are tested at 10 micromolar. Please see reference 11 for details.
This assay measures increases of intracellular calcium in transfected HEK 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:
There are no active compounds in this assay. The PubChem Score range for inactive compounds is 0-0.
List of Reagents:
TRP HEK293 cell lines (provided by Prof. Stefan Heller)
Categorized Comment - additional comments and annotations
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