FIGURE 6.

Insertion of GABA_ARs is modulated by Ca²⁺ influx through L-type VGCCs. A, images of pHluorin fluorescence and Alexa 594-conjugated α-Bgt staining at 5 min of insertion in the presence or absence of nifedipine (Nif; 10 μm) as indicated. Hippocampal neurons expressing ^BBSβ3 (18–21 DIV) were treated with or without nifedipine (10 μm) for 24 h. Neurons were then incubated with 10 μg/ml unlabeled α-Bgt to block existing cell-surface ^BBSβ3, washed, incubated for 5 min with 1 μg/ml Alexa 594-conjugated α-Bgt to label newly inserted ^BBSβ3, and fixed. The boxed areas in the upper right-hand corners show pHluorin fluorescence. The rectangles in the left panels (Alexa 594-conjugated α-Bgt staining) are enlarged in the right panels. B, increase in ^BBSβ3 insertion over time with or without nifedipine (10 μm). The assay was performed as described for A. C, graph showing quantification of Alexa 594-conjugated α-Bgt fluorescence intensity at 5 min of insertion for control (Ctrl) and nifedipine (10 μm)-treated neurons. Data represent the mean ± S.E. percentage of control ^BBSβ3 values. *, significantly different from the control (p < 0.01; t test; n = six to eight neurons from two independent cultures).