Figure 5.

GABA_BR activation leads to a reduction in KCC2 at the membrane surface. A, GABA_BR activation leads to a reduction in the fraction of both monomeric and dimeric forms of KCC2 at the membrane surface. Rat organotypic hippocampal slices (P7 + 7–14 DIV) were exposed to normal ACSF (Control) or ACSF containing SKF97541 for 20 min (SKF, 5 μm), and then biotinylated to label proteins at the cell surface. Cell homogenates of total protein (T) and streptavidin-purified cell surface proteins (S) were probed on Western blots with anti-KCC2 antibodies (top). Lack of β-tubulin staining in surface samples confirmed that the cell membranes remained intact (bottom). B, SKF97541 treatment also led to a concomitant reduction in surface levels of GABA_BR1 relative to controls. C, Neither surface-bound NKCC1 nor the surface levels of the transferrin receptor changed following GABA_BR activation. D, Summary plot of the effect of GABA_BR activation upon proteins at the membrane surface after 20 min 5 μm SKF97541 treatment. Surface expression was quantified as the ratio of surface to total protein, normalized to control slices that were examined in parallel. SKF97541 treatment resulted in a significant reduction in the surface ratio for KCC2 (monomer, **p = 0.002; dimer, *p = 0.048; all, **p = 0.003; n = 14, t test) and GABA_BR1 (all, *p = 0.046, n = 9, t test). No effect was observed in the surface/total ratio for NKCC1 (p = 0.4, n = 13, t test) or the transferrin receptor (TfR; p = 0.79, n = 8, t test). E, The association between KCC2 and GABA_BR remains following SKF97541 treatment. Rat organotypic hippocampal slices were solubilized and subjected to coimmunoprecipitation analysis using GABA_BR1 as the precipitating antibody. The amount of KCC2 in GABA_BR1 complexes (top), normalized by the amount of GABA_BR1 protein precipitated (bottom), did not change following SKF97541 treatment (p = 0.44, n = 3, t test).