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_PATCH-CLAMP LATE STAGE Counterscreen

Name: Late stage counterscreen results from the probe development effort to identify selective agonists of the Transient Receptor Potential Channels 3 (TRPML3): TRPN1 patch clamp 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, TRPN1, NompC, counterscreen, TRP, N1, agonist, agonism, activator, activation, activate, patch clamp, whole cell, capacitance, current, recordings, voltage, cells, cell-based, counterscreen, assay provider, assay, Scripps, Scripps Florida, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.

The purpose of these assays is to determine if compounds identified as TRPML3 agonists were nonselective due to activation of the TRPN1 ion channel, as measured by an increase in current recordings. Compounds were tested at 10 micromolar. Please see reference 11 for details.

Protocol Summary:

Whole-cell currents were recorded with an Alembic Instruments VE-2 amplifier with 100% series resistance compensation, and acquired with JClamp software. The standard bath solution contained 138 mM NaCl, 5.4 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM HEPES, and 10 mM d-glucose, adjusted to pH 7.4 with NaOH. The standard pipette solution contained 140 mM CsCl, 10 mM HEPES, 3 mM ATP-Na, 1 mM BAPTA, and 2 mM MgCl2, adjusted to pH 7.2. 2-Aminoethyl-diphenyl borate (100 micromolar) was included in the bath solution to block gap junctions and had no effect on the expressed channels. The data collected were responses to 10 ms voltage steps (holding potential, +10 mV) between −200 mV and +100 mV in 20 mV incremental steps, normalized by cell capacitance (pF) (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 active compounds.

List of Reagents:

TRPN1 HEK293 cell line (provided by Prof. Stefan Heller)

This assay was performed by the assay provider.

Grant Number: 1 R03 MH083077-01