FIGURE 6.

Kinase inhibition prevents the Ca²⁺-dependent phosphorylation of STX3B at T14 in rod bipolar cell synaptic terminals. (A) Confocal images of synaptic terminals of isolated rod bipolar cells, identified by their characteristic morphology and PKC immunoreactivity (green), demonstrate an increase in pSTX3 immunoreactivity (red) under conditions of elevated intraterminal Ca²⁺ (“High Ca²⁺”) when compared to those incubated in a nominally 0 Ca²⁺ external solution (“Low Ca²⁺”). Treatment with staurosporine (100 nM; lower panel) blocked the expected Ca²⁺-evoked increase in pSTX3 immunoreactivity. Scale bar = 5 μm. (B) Quantification of the pSTX3/STX3 ratio in rod bipolar cell terminals shows that elevated intraterminal Ca²⁺ (“High”) significantly increased the pSTX3/STX3 ratio above that of cells bathed in external solution containing no added Ca²⁺ (“Low”; p < 0.0001, n = 4,4 mice). Staurosporine not only blocked this Ca²⁺-evoked rise in the pSTX3/STX3 ratio (p < 0.0001, n = 4,4 mice), it lowered the ratio of the low Ca²⁺ control group (p = 0.0366, n = 4,3 mice). (C) In parallel experiments, treatment with staurosporine suppressed the Ca²⁺-stimulated increase in pCaMKII immunolabeling (p < 0.0001, n = 3,3 mice). *Indicates p < 0.05, ***indicates p < 0.001, and ****indicates p < 0.0001.