Identification of Novel Modulators of Cl- dependent Transport Process via HTS: Rubidium Flux
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, more ..
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Depositor Specified Assays
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 assay was to test lead compounds identified from thallium flux assays in radioactive rubidium [86Rb] flux assays for inhibition of KCC2 and NKCC1 function in HEK293 cells.
For KCC2 measurements through [86Rb] uptake assays, KCC2-overexpressing HEK293 cells were briefly trypsinized from 10-cm dishes and plated for 2 hours on 35-mm dishes coated with poly-L-Lysine (0.1 mg/ml, Sigma). Cells were washed once and pre-incubated for 15 min with 1 ml hyposmotic saline solution containing in mM: 120 NaCl, 5 KCl, 2 CaCl2, 0.8 MgSO4, 5 HEPES, 5 glucose, 100 uM ouabain, 500 uM N-ethylmaleimide, pH 7.4 with HCl (260-270 mOsm). After the preincubation period, the medium was aspirated and replaced with 1 ml of similar solution without NEM, but containing 1 uCi/ml [86Rb]. After 15 min uptake, the solution was aspirated and the cells were washed thrice with ice-cold solution. The cells were then lysed for 1 hour with 500 ul 0.25N NaOH and then neutralized with 250 ul glacial acetic acid. A 300 ul aliquot was then added to 5 ml liquid scintillation fluid for counting and a 30 ul aliquot was used for protein assay. A 5 ul aliquot of the uptake solution was also counted as standard. Uptakes are expressed in pmole K+/mg protein/min, and the furosemide-sensitive uptake (KCC mediated flux) is calculated as the difference between the uptake measured in absence of furosemide and the flux obtained in the presence of 2 mM furosemide. Each experimental condition was measured in triplicate.
For NKCC1, [86Rb] uptake experiments were performed with naive HEK293 cells incubated in a hyperosmotic saline. The solution contained in mM: 120 NaCl, 5 KCl, 2 CaCl2, 0.8 MgSO4, 5 HEPES, 5 glucose, 100 uM ouabain, 110 mM sucrose, pH 7.4 with HCl (370-380 mOsm). Experiment was performed as described for KCC2.
Fluxes were expressed as percent of control and fitted using a sigmoidal dose-response (non linear) regression (GraphPad Prism version 3.0).
Note that for KCC2, we used N-ethylmaleimide pre-treatment. The alkylating agent not only stimulates K-Cl cotransport, but abrogates the function of the native Na-K-2Cl cotransporter. Under the above conditions, furosemide and bumetanide had IC50s of 455 and 655 uM, respectively.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)