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BioAssay: AID 2694

Late stage results from the probe development effort to identify selective agonists of the Transient Receptor Potential Channels 3 (TRPML3): TRPML3 patch clamp assay

Name: Late stage results from the probe development effort to identify selective agonists of the Transient Receptor Potential Channels 3 (TRPML3): TRPML3 patch clamp assay. ..more
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 Tested Compounds
 Tested Compounds
All(3)
 
 
Active(3)
 
 
 Tested Substances
 Tested Substances
All(3)
 
 
Active(3)
 
 
AID: 2694
Data Source: The Scripps Research Institute Molecular Screening Center (TRPML3-SELECTIVE_AG_PATCH-CLAMP LATE STAGE)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-03-26

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 3
Depositor Specified Assays
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AIDNameTypeProbeComment
1424Primary cell-based high-throughput screening assay to identify agonists of the transient receptor potential channel N1 (TRPN1)screening 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 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 Counterscreen Assay (TRPN1 agonists, 3X%ACT)
1526Confirmation cell-based high-throughput screening assay for agonists of the transient receptor potential channel ML3 (TRPML3)screening 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 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 Dose Response Counterscreen (TRPN1 agonists, 3X EC50)
1809Summary of probe development efforts to identify agonists of the transient receptor potential channel ML3 (TRPML3)summary2 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 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 Dose Response Counterscreen (TRPN1 agonists, 3X EC50)
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-SELECTIVE_AG_PATCH-CLAMP LATE STAGE

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

Keywords:

Late stage, probes, selective, TRPML3, TRP, cation channel, mucolipin 3, MCOLN3, deafness, HEK 293, 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.
Protocol
The purpose of these assays is to determine if test compounds can increase current recordings in TRPML3 ion channels. 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 μM) 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 inactive compounds.

List of Reagents:

TRPML3 HEK293 cell line (provided by Prof. Stefan Heller)
Comment
Probes were identified. This assay was performed by the assay provider.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Ion Channel Current (Average) [nA/pF, -80 mV] (10μM**)The ion channel current 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). Average of three measurements.Float
2Ion Channel Current (StdErr) [nA/pF, -80 mV]Standard Error of the Ion Channel Current measurement.Float
3Probe Molecule OutcomeIndicates whether the compound is assigned to be a probe or not. One of "Probe", "Active" or "Inactive".String

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

Data Table (Concise)
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