KCC2/SLC12A5 Antibody (S1-12) NBP2-59337 from Novus Biologicals, a Bio-Techne Brand

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Frontiers in cell and developmental biology
January 01 2022

Fig 1: Discovery of KCC2 Potentiators by High-throughput Screening. (A) Cl- flux assay schematic and data normalization. KCC2 activity is approximated by measuring intracellular Cl- using HEK-293 cells overexpressing KCC2 and the Cl- sensor SuperClomeleon. Data are normalized as a YFP/CFP ratio, such that an increase in KCC2 activity registers as an increase in value. (B,C) Calculation of Z' for the Cl- flux assay. (B) Vehicle control subtracted and averaged traces from the Cl- flux assay used for Z' calculation. Cells were treated with either the indirect KCC2 potentiator WNK463 or DMSO control. Error bars represent standard deviation of the mean (SD). (C) Percent Area Under the Curve (AUC) of Cl- flux assay traces in (B). The mean AUC (solid line) and three x SD away from mean AUC (dotted line) are displayed for each treatment (n = 192). (D) Results of screening approximately 23,000 unique compounds at a concentration of 10 µM in the Cl- flux assay. Data are presented as Z-scores relative to vehicle control values on each plate. A Z-score of 3 (dotted line) was used as a cut off for separating potentiator hits (gold) from uncategorized compounds (gray). All experiments were performed at 37°C.
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Frontiers in cell and developmental biology
January 01 2022

Fig 2: Development of VU0916219, a KCC2 potentiator with improved activity. Experiments conducted at 37°C, using the Cl- Flux Assay in HEK-293 cells overexpressing KCC2. Data are normalized to maximum VU0500469 activity being 100%. (A) Structures of the most potent and efficacious VU0500469 analogs. (B) Comparison of VU0500469 dose-response curve to the most potent (left panel) and efficacious (right panel) compounds. (C) Structure of VU0916219. (D) Comparison of VU0916219 to the most efficacious compound (left panel), and VU0500469 (right panel). (n = 6), error bars represent SD.
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Frontiers in cell and developmental biology
January 01 2022

Fig 3: VU0500469 potentiates KCC2 by a unique mechanism. All experiments conducted using the Cl- flux assay in HEK-293 cells overexpressing KCC2 at 37°C, unless otherwise noted. (A) Schematic depicting investigated small molecule targets known to regulate Cl- homeostasis. (B) Co-treatment of modulators of Cl- homeostasis from (A) with or without VU0500469 (n = 16). (C) Left: representative Western blot of biotinylated surface fraction from cells treated with vehicle (DMSO), VU0916219 (VU219), or the positive control phorbol 12-myristate 13-acetate (PMA). Band intensity was normalized to Transferrin Receptor (TfR) levels. O, oligomeric KCC2, M, monomeric KCC2. Right: quantification of Surface KCC2/Total KCC2 normalized to TfR levels. (D) Left: representative Western blot of total protein fraction from cells treated as in (C). Right: quantification of total KCC2 normalized to TfR levels. (n = 4) Error bars represent SD. Statistical significance calculated by Mann–Whitney U test (B) or paired t-test (C,D): * = p < 0.05, *** = p < 0.001.
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Frontiers in cell and developmental biology
January 01 2022

Fig 4: VU0500469 inhibits seizure-like, synchronous Ca2+ oscillations in cultured rat cortical neurons. (A) Cartoon description of the synchronous Ca2+ oscillation assay, which uses neuronal Ca2+ oscillations from high density neuronal-glial cocultures to model epileptiform activity. (B,C) Validation of the synchronous neuronal Ca2+ oscillation assay’s ability to report anti-epileptic and KCC2-dependent activity. (B) Example Ca2+ oscillation trace treated with the antiepileptic drug carbamazepine (CBZ). Segments before and after compound addition are highlighted to better show neuronal Ca2+ oscillations in the absence and presence of compound, respectively. The time point of compound addition denoted by the red line. Oscillations recognized by a spike counting algorithm are marked by a red “X.” (C) Quantified % pre-addition oscillation rate for CBZ, the GABAA positive allosteric modulator diazepam, vehicle control, the convulsant 4-AP, and the KCC2 inhibitor VU0463271 (n = 3). (D–F) VU500469 dose-dependently prevents seizure-like Ca2+ oscillations. (D) VU0500469 structure. (E) Example trace treated with 20 µM VU0500469, annotated as in (B). (F) Dose-dependent effect of VU0500469 on Ca2+ oscillation rate (n = 10). (G–I) VU0500469, a structurally related analog of VU0500469 without significant activity in the Cl- flux assay, does not affect seizure-like Ca2+ oscillations. (G) VU0500469 structure. (H) Example trace treated with 20 µM VU0500469, presented as in (B). (I) Dose-dependent effect of VU0500469 on % pre-addition oscillation rate. Error bars represent SD. (n = 10). Statistical significance calculated by one sample t-test (6B, DMSO v. diazepam) and Welch’s t-test for all other samples: ** = p < 0.01, *** = p < 0.001.
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Frontiers in cell and developmental biology
January 01 2022

Fig 5: VU0500469 (VU469) potentiates KCC2-mediated Cl- transport across mechanistically distinct assays of KCC2 activity in HEK-293 cells. (A,B) Effect of 15 µM VU0500469 on Cl- flux in the presence or absence of KCC2 overexpression (n > 9). (A) Cl- Flux Assay in cells overexpressing KCC2. Left: AUC of the YFP/CFP Cl- efflux ratio. Right: Vehicle control subtracted and averaged traces of the YFP/CFP Cl- efflux ratio. Dotted line represents time of compound addition. (B) Cl- flux assay in cells lacking KCC2 overexpression. (C,D) Effect of 15 µM VU0500469 on Tl+ influx in the presence or absence of KCC2 overexpression (n > 20). (C) Tl+ influx in cells overexpressing KCC2. Left: maximum fold increase in Tl+-sensitive dye fluorescence. Right: fold increase in Tl+-sensitive dye fluorescence over time. Dotted line represents time point of Tl+ addition. (D) Tl+ influx in cells lacking KCC2 overexpression. (E) Effect of 30 µM VU0500469 on K+ influx in cells overexpressing KCC2 in the absence or presence of KCC2 inhibitor ML077 (n = 3). (F) VU0500469 structure. All experiments conducted at 37°C. Error bars represent SD. Statistical significance calculated by Welch’s t-test (E) and Mann–Whitney U test (A–D): * = p < 0.05, *** = p < 0.001, ns = p > 0.05.