FIGURE 3.

Ca²⁺ influx through voltage-gated Ca²⁺ channels is necessary for glutamate-induced down-regulation of GABA_B receptors. A, removal of extracellular Ca²⁺ using EGTA completely inhibited glutamate- and AMPA-induced down-regulation of GABA_B receptors. Cells were treated with either 50 μm glutamate, 100 μm AMPA, or 5 mm EGTA or with the indicated combinations for 90 min and tested for GABA_B receptor levels using the in-cell Western assay. Means ± S.D.; n = 25–32 cultures from four preparations; ***, p < 0.001, one-way ANOVA; Bonferoni post test. B, inhibition of L-type Ca²⁺ channels but not P/Q-type and N-type channels prevents down-regulation of GABA_B receptors. Neurons were incubated for 90 min either with 50 μm glutamate, 50 μm glutamate +100 μm NASPM (blocker of Ca²⁺-permeable AMPA receptors) +100 μm nifedipine (L-type Ca²⁺ channel blocker), +2 μm ω-conotoxin MVIIC (P/Q and N-type Ca²⁺ channel blocker), +0.5 μm ω-Agatoxin TK (P/Q-type Ca²⁺ channel blocker), or +3 μm ω-conotoxin GVIA (N-type Ca²⁺ channel blocker) and subjected to the in-cell Western assay for determination of GABA_B receptor levels. Mean ± S.D., n = 16–32 cultures from two to four preparations; n.s. = p > 0.05, ***, p < 0.001, one-way ANOVA; Bonferoni post test.