Sequence: electroneutral potassium-chloride cotransporter KCC2 [Homo sapiens]

Gene:SLC12A5     Conserved Domain     Related Protein 3D Structures

Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters
Assay Provider: Eric Delpire
Assay Provider Affliation: Vanderbilt University
Grant Title: Identification of Novel Modulators of Cl- dependent Transport Process via HTS
Grant Number: R21NS053658-01

Cation-chloride cotransporters such as K-Cl cotransport and Na-K-2Cl cotransport play major roles in a variety of physiological settings, including the modulation of GABAergic synaptic transmission. For instance, KCC2, a neuronal-specific K-Cl cotransporter is up-regulated in the brain during postnatal development, and is responsible for lowering the intracellular Cl- concentration in neurons, thus promoting GABA inhibition. Reduction in KCC2 expression results in brain hyperexcitability, as demonstrated by animal models. Furthermore, KCC2 expression is decreased in brain tissue isolated from epileptic patients.

There are very few pharmacological agents that affect K-Cl cotransporters. First, there are no specific inhibitors of K-Cl cotransporters. Furosemide is mostly used to inhibit K-Cl cotransporter function, but the diuretic is not very potent and is not specific as it inhibits the Na-K-2Cl cotransporter (diuretic effect), many Cl- channels including the GABAA receptor. Finding new inhibitors will provide important tools for the study of KCC2 in modulating inhibitory neurotransmission. Second, there are also no compounds known to activate K-Cl cotransporter, except for N-ethylmaleimide, which affects many cellular processes as an unspecific alkylating agent. Finding a specific agent that increase KCC2 function would potentially have therapeutic value, as increased KCC2 function reduces susceptibility to epileptic seizures.

The purpose of this screen was to test compounds from the Molecular Libraries Small Molecule Repository (MLSMR) at a single concentration in a cell-based kinetic flux assay for modulators of the cation-chloride cotransporter, KCC2.

METHOD:
1. Human embryonic kidney cells stably transfected with KCC2 were plated at 20,000 cells/well in Dulbecco's modified medium (DMEM), 41.6% F12 (Gibco catalog 11320-033) in 384 well plate, black, clear bottom, poly-D-lysine coated (Greiner catalog 781946).
2. Cells were incubated overnight at 37 degrees C in 5%CO2.
3. Cells were loaded with 0.5 micromolar FluoZin2-AM dye (Invitrogen catalog number F24189) in assay buffer (Hanks Buffered Salt Solution, 20 mM HEPES, 0.2 mM oubain) for 48 minutes.
4. Dye was removed and the plate imaged using the Hamamatsu FDSS kinetic plate reader equipped with 480 nanometer excitation and 540 nanometer emission filters.
5. Compounds in DMSO were added to a final concentration of 10 micromolar (0.1% DMSO final concentration).
6. Thallium buffer stimulus (125 mM sodium bicarbonate, 12 mM thallium sulfate, 1 mM magnesium sulfate, 1.8 mM calcium sulfate, 5 mM glucose, 10 mM HEPES, pH 7.3.) was added and images collected at 1 Hz.
7. Assay buffer containing DMSO (0.1% final concentration) was used as the negative control and 2 mM bumetanide was used as the positive control on each plate.

DATA PROCESSING:
1. The raw fluorescence intensities were divided by the initial fluorescence, and the slope from 10 to 20 seconds after thallium addition was calculated and labeled 'Value.'
2. Bscore [1] was calculated using 'Value' normalized for row and column effects on a per-plate basis.
3. 'Value' and 'BScore' were treated as Gaussian distributions.
4. Compounds selected with 'Score' of '100' and 'Outcome' of 'Active' had values that differed from the mean sample distribution at 99.7% confidence level after each of 2 rounds of filtering.
5. All calculations were done on a per-plate basis using Pipeline Pilot with the R statistics package.

References:
1. Malo N, Hanley JA, Cerquozzi S, Pelletier J, Nadon R. Statistical practice in high-throughput screening data analysis. Nat Biotechnol. 2006 Feb; 24(2):167-75.

These data reflect the observations from primary screening where the number of independent observations or sample size is one (n = 1). Possible artifacts include, but are not limited to, dust in or on the microtiter plate, compounds that fluoresce, and compounds or conditions that cause a change in thallium levels.

Grant Number: R21NS053658-01