Sequence: cholinergic receptor, muscarinic 4 [Mus musculus]

Gene:CHRM4     Conserved Domain     Related Protein 3D Structures

Assay Provider: Colleen Niswender
Assay Provider Affliation: Vanderbilt University
Grant Title: Discovery of Novel Allosteric Agonists of the M4 Muscarinic Receptor
Grant Number: 1 X01 MH077607-1

The focus of this screening campaign was to identify highly selective small molecules that act as allosteric agonists of the M4 muscarinic receptor. The assay utilized thallium influx through G-protein Inwardly Rectifying K+ (GIRK) channels as a measure of M4 activation. Compounds were tested at 10uM final concentration against Human Embryonic Kidney (HEK) 293 cells stably expressing cDNAs for GIRK 1, GIRK 2, and the M4 muscarinic receptor. The Hamamatsu FDSS 6000 plate reader was used to collect kinetic fluorescence intensities during treatment with the test compound. The muscarinic agonist carbachol was the positive control, and DMSO, the compound vehicle, was used as the negative control.

Assay Protocol:
M4/GIRK expressing HEK293 cells were plated at 15,000 cells per well in Dulbecco's modified medium (DMEM) containing 10% dialyzed fetal bovine serum (FBS), 100units/mL penicillin/streptomycin, and 20mM HEPES. 384-well, black walled, clear bottom, poly-D-lysine coated plates were used. After overnight incubation at 37degreesC in the presence of 5% CO2, the medium was removed and BTC-AM (Molecular Probes) dye in assay buffer (Hanks Balanced Salt Solution (HBSS)) was added. After 1hour, the dye was replaced with HBSS. Test compounds were prepared in thallium assay buffer (125mM sodium gluconate, 1mM magnesium sulfate, 1.8mM calcium gluconate, 5mM glucose, 12mM thallium sulfate, 10mM HEPES, pH 7.3). The compounds were added to the cell plate by the Hamamatsu FDSS. Each cell plate was imaged at 1Hz for 10seconds, 10uL of test compound was added, and the cell plate was imaged at 1Hz for 2minutes total sampling time.

Data Processing:
Each kinetic trace was normalized to the initial fluorescence intensity to correct for dye loading of the cells. The ratios were used to calculate the slope of the response after compound addition (time 15 to 24 seconds). Slope values used resulted from a linear fit with 30 iterations or with a chi squared value less than or equal to 0.0001. The slope for each test compound response was compared to the vehicle control sample, DMSO. Compounds selected as 'Outcome' = 'Active' and 'Score' = '100' had slope values greater than the 'Cutoff_Value' calculated as the mean of the vehicle control (DMSO) plus three times the standard deviations.

These data are from experiments where the number of independent measurements is one (n=1). Possible artifacts of this assay include, but are not limited to dust in or on the microtiter plate, compounds that fluoresce, compounds that interact with BTC-AM, and compounds that cause nonspecific influx of thallium.