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BioAssay: AID 504425

Mode of action assay-Specificity dose response assay for the identification of selective inhibitors of T-type calcium subunit Cav3.2 in the Cav3.3 expressing cell line on automated patch clamp

Name: Mode of action assay-Specificity dose response assay for the identification of selective inhibitors of T-type calcium subunit Cav3.2 in the Cav3.3 expressing cell line on automated patch clamp ..more
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 Tested Compounds
 Tested Compounds
All(4)
 
 
Active(2)
 
 
Inactive(2)
 
 
 Tested Substances
 Tested Substances
All(4)
 
 
Active(2)
 
 
Inactive(2)
 
 
AID: 504425
Data Source: Johns Hopkins Ion Channel Center (JHICC_Cav3.3_CRC_IWS)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-03-08
Hold-until Date: 2012-03-06
Modify Date: 2012-03-06

Data Table ( Complete ):           Active    All
Target
Sequence: voltage-dependent calcium channel T-type alpha 1I subunit [Homo sapiens]
Description ..   

Gene:CACNA1I     Conserved Domain     Related Protein 3D Structures     More BioActivity Data..
BioActive Compounds: 2
Depositor Specified Assays
AIDNameTypeComment
463087Inhibitors of Cav3 T-type Calcium Channelssummary
449739Inhibitors of Cav3 T-type Calcium Channels: Primary Screenscreening
489005Inhibitors of T-Type Calcium Channelconfirmatory
493041Inhibitors of T-Type Calcium Channels (SynthLib3)confirmatory
504584Inhibitors of T-Type Calcium Channels (Ancillary Pharm)other
504619Inhibitors of T-Type Calcium Channels (Cav3.2 HEK whole cell CRC)confirmatory
504628Inhibitors of T-Type Calcium Channels (Cav3.2 HEK whole cell)other
504579Inhibitors of T-Type Calcium Channels (rat DRG neuron currents)other
Description:
Name: Mode of action assay-Specificity dose response assay for the identification of selective inhibitors of T-type calcium subunit Cav3.2 in the Cav3.3 expressing cell line on automated patch clamp
Data Source: Johns Hopkins Ion Channel Center (JHICC_Cav3.3_CRC_IWS)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed

Source (MLPCN Center Name): Johns Hopkins Ion Channel Center (JHICC)
Center Affiliation: Johns Hopkins University, School of Medicine
Screening Center PI: Min Li, Ph.D.
Assay Provider: Xinmin Xie, Ph.D.
Affiliation: Bioscience Division, SRI International, Menlo Park, CA
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: NS050771-01
Grant Proposal PI: Xinmin Xie, Ph.D.
Assay Implementation: Haibo Yu Ph.D., Kaiping Xu M.S., Meng Wu Ph.D., Owen McManus Ph.D.

Description:

T-type Ca2+ channels are also called low voltage-activated channels because they open at voltages near the resting membrane potential of most cells. In many types of neurons, Ca2+ influx through T-type channels triggers low-threshold spikes, which in turn trigger a burst of action potentials mediated by Na+ channels (1). Burst firing is thought to play an important role in the synchronized activity of the thalamus observed in absence epilepsy, and also in a wider range of neurological disorders characterized by thalamocortical dysrhythmia (2). Prominent T-currents are also observed in dorsal root ganglion neurons, with subsets of nociceptors expressing more T-current than high voltage-activated Ca2+ currents (3). Considerable evidence supports the notion that a T-channel antagonist would be a useful drug for the treatment of pain and epilepsy (4).

1. Perez-Reyes, E: Molecular physiology of low-voltage-activated T-type calcium channels. Physiol. Rev. 2003; 83: 117-161.
2. Llinas, R R, Ribary, U, Jeanmonod, D, Kronberg, E, and Mitra, P P: Thalamocortical dysrhythmia: A neurological and neuropsychiatric syndrome characterized by magnetoencephalography. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 15222-15227.
3. Nelson, M T, Joksovic, P M, Perez-Reyes, E, and Todorovic, S M: The endogenous redox agent L-cysteine induces T-type Ca2+ channel-dependent sensitization of a novel subpopulation of rat peripheral nociceptors. J. Neurosci. 2005; 25: 8766-8775.
4. Nelson, M, Todorovic, S, and Perez-Reyes, E: The role of T-type calcium channels in epilepsy and pain. Curr Pharm Des 2006; 12: 2189-2197.


Principle of the assay
Patch clamp is gold standard to measure channel activities. The purpose of the assay is to examine the non-specific effect of Cav3.2 inhibitors in the Cav3.3 expressing cell line. This assay employs automated patch clamp (Ionworks Quattro) to investigate the current response of Cav3.3-HEK293(LT11) elicited by voltage clamp protocols in the presence or absence of test compounds. Compounds were tested in quadruplicates at 8-point concentrations with 3-fold dilutions.

Keywords:

T-type calcium channel,Cav3.2, Cav3.3,Inhibitor, Antagonist, Concentration Response Curve, JHICC, Johns Hopkins, Molecular Libraries Probe Production Centers Network, MLPCN.
Protocol
Protocol for automated patch clamp on Cav3.3-HEK293 cells with voltage clamp

The HEK293 cells stably expressing CaV3.3 were freshly dislodged from flasks and dispensed into a 384-well population patch clamp (PPC) plate. The cells plating density was 7,000 cells/well suspended in the extracellular solution, composed of (in mM): 137 NaCl, 4 KCl, 1 MgCl2, 1.8 CaCl2, 10 HEPES, and 10 glucose, pH 7.4 adjusted with NaOH .
After dispensing, seal resistance of cells was measured for each well and cells were perforated by incubation with 50microg/mL amphotericin B (Sigma, St. Louis, MO), which was dissolved in the internal solution composed of (in mM): 40 KCl, 100 K-Gluconate, 1 MgCl2, 5 HEPES, 2 mM CaCl2 pH 7.2 adjusted with KOH. The channel activity was then measured with the recording protocol as followings. Leak currents were linear subtracted extrapolating the current elicited by a 100-ms step to -110 mV from a holding potential of -100 mV. During the voltage pulse protocol, cells were held at -100 mV, followed by depolarization step to -30mV for 1 s, and then back to -100mV. The currents were measured at the peak of the inward currents before and after the application of compounds for 3 min. Only cells with a current amplitude more than 200 pA at -30 mV and a seal resistance >30 MOhms were included in the data analysis.
Compound effects were assessed by the percentage changes in the inward currents, which were calculated by dividing the difference of current amplitude between pre- and post-compound recording by the respective pre-compound currents in the same well.

1.Outcome assignment
If the test compound causes inhibition effect on Cav3.3 at any concentrations tested and the dose response is generated, the compound is considered to be active.
If the test compound does not cause inhibition effect on Cav3.3 at any concentrations tested or a dose response is not generated, the compound is designated as inactive.
2.Score assignment
An inactive test compound is assigned the score of 0.
An active test compound is assigned the score of 100.
Comment
Possible artifacts of this assay may include, but are not limited to: unintended chemicals or dust in or under the wells of the microtiter plate, or compounds that quench or emit light or 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.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1percentage at 14nM (0.014μM**)% percentage for Cav3.3 at the specified concentrationFloat
2SD at 14nMStandard Deviation of measurement for % percentage at the specified concentrationFloat
3percentage at 41nM (0.041μM**)% percentage for Cav3.3 at the specified concentrationFloat
4SD at 41nMStandard Deviation of measurement for % percentage at the specified concentrationFloat
5percentage at 120nM (0.12μM**)% percentage for Cav3.3 at the specified concentrationFloat
6SD at 120nMStandard Deviation of measurement for % percentage at the specified concentrationFloat
7percentage at 370nM (0.37μM**)% percentage for Cav3.3 at the specified concentrationFloat
8SD at 370nMStandard Deviation of measurement for % percentage at the specified concentrationFloat
9percentage at 1.1uM (1.1μM**)% percentage for Cav3.3 at the specified concentrationFloat
10SD at 1.1uMStandard Deviation of measurement for % percentage at the specified concentrationFloat
11percentage at 3.3uM (3.3μM**)% percentage for Cav3.3 at the specified concentrationFloat
12SD at 3.3uMStandard Deviation of measurement for % percentage at the specified concentrationFloat
13percentage at 10uM (10μM**)% percentage for Cav3.3 at the specified concentrationFloat
14SD at 10uMStandard Deviation of measurement for % percentage at the specified concentrationFloat
15percentage at 30uM (30μM**)% percentage for Cav3.3 at the specified concentrationFloat
16SD at 30uMStandard Deviation of measurement for % percentage at the specified concentrationFloat
17EC50*mean EC50 of compound on Cav3.3FloatμM
18EC50 SDStandard deviation of mean of EC50FloatμM
19Nnumber of repeats for EC50Integer
20HillHill constantFloat
21Hill SDStandard deviation of Hill constantFloat

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 DA027716-01

Data Table (Concise)
Classification
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