Dopamine-stimulated dephosphorylation of connexin 36 mediates AII amacrine cell uncoupling

Abstract

Gap junction proteins form the substrate for electrical coupling between neurons. These electrical synapses are widespread in the CNS and serve a variety of important functions. In the retina, connexin 36 (Cx36) gap junctions couple AII amacrine cells and are a requisite component of the high-sensitivity rod photoreceptor pathway. AII amacrine cell coupling strength is dynamically regulated by background light intensity, and uncoupling is thought to be mediated by dopamine signaling via D(1)-like receptors. One proposed mechanism for this uncoupling involves dopamine-stimulated phosphorylation of Cx36 at regulatory sites, mediated by protein kinase A. Here we provide evidence against this hypothesis and demonstrate a direct relationship between Cx36 phosphorylation and AII amacrine cell coupling strength. Dopamine receptor-driven uncoupling of the AII network results from protein kinase A activation of protein phosphatase 2A and subsequent dephosphorylation of Cx36. Protein phosphatase 1 activity negatively regulates this pathway. We also find that Cx36 gap junctions can exist in widely different phosphorylation states within a single neuron, implying that coupling is controlled at the level of individual gap junctions by locally assembled signaling complexes. This kind of synapse-by-synapse plasticity allows for precise control of neuronal coupling, as well as cell-type-specific responses dependent on the identity of the signaling complexes assembled.

Figure 1.

AII amacrine cell coupling is directly related to Cx36 phosphorylation at Ser293. A–C, Neurobiotin tracer coupling between AII amacrine cells is modulated by dopamine D₁R signaling. D₁R activation [B; SKF38393 (SKF), 10 μm] reduced the extent of Neurobiotin diffusion relative to control (A). D₁R antagonism [C; SCH23390 (SCH), 100 μm] increased tracer diffusion. Images are mini-stacks (2 μm in z-depth) focused on the somas of the AII amacrine cells. Yellow boxes highlight areas shown in D–I (at different focal depth). D–F, Cx36 gap junctions, labeled with mCx36 antibody (red) and Ser293-P antibody (green), on and around the dendrites of the injected AII amacrine cell, labeled with fluorophore-conjugated streptavidin (blue). The Cx36 gap junctions not on the injected cell are primarily on other AII amacrine cells (see Materials and Methods). Arrowheads identify prominent Cx36 gap junctions on the injected cells. G–I, Phosphorylation of Cx36 at Ser293, a site known to regulate coupling through Cx36 gap junctions (Ouyang et al., 2005), is also modulated by dopamine D₁R signaling. Arrowheads identify the locations of the same Cx36 gap junctions identified in D–F. D₁R activation (H; SKF38393, 10 μm) reduced Ser293-P labeling relative to control (G). D₁R antagonism (I; SCH23390, 100 μm) increased Ser293-P labeling. J, Quantification of the relationship between AII amacrine cell coupling and Cx36 phosphorylation at Ser293. The mean ratio of Ser293-P intensity to mCx36 intensity (across all Cx36 gap junctions in 3 images per injection) is plotted against the diffusion coefficient for Neurobiotin tracer transfer calculated for each injected AII amacrine cell network. The strong correlation of the data (r ² = 0.86) indicates a direct relationship between AII amacrine cell coupling and Cx36 phosphorylation at Ser293. Images are 2-μm-deep stacks. Scale bars: C, 50 μm; F, 5 μm.

Figure 2.

PKA mediates D₁R-dependent dephosphorylation of Cx36 at Ser293 in AII amacrine cells. A, B, Under control conditions, Ser293-P (green) labeling of Cx36 gap junctions (mCx36, red) on AII amacrine cells (calretinin, blue) was heterogeneous. Annotated arrowheads indicate pairs of Cx36 plaques along single dendrites that are in different phosphorylation states. The close proximity of Cx36 plaques with widely varying Ser293-P labeling implies that regulation is locally controlled at individual plaques. C, D, D₁R activation (SKF38393, 100 μm) greatly diminished Ser293-P labeling. E, F, Inhibition of PKA [Rp-8-CPT-cAMPS (Rp), 20 μm] attenuated the dramatic reduction in Ser293-P labeling caused by SKF38393 (SKF; 100 μm). G, H, Antagonism of D₁Rs [SCH23390 (SCH), 100 μm] increased Ser293-P labeling. I, Summary of data shows that inhibition of PKA significantly suppressed the reduction in Ser293 phosphorylation caused by D₁R activation. J, Summary of data shows that changes in the percentage of Cx36 plaques that show detectable Ser293-P labeling follows the same pattern established for relative Ser293-P measurements in I. Error bars are SEM, n = 6. *p < 0.05, **p < 0.01. Images are 1-μm-deep stacks. Scale bar: H, 10 μm.

Figure 3.

Phosphorylation of Ser293 on Cx36 does not alter trafficking or distribution of the protein. A, Conditions that altered Cx36 phosphorylation at Ser293 [D₁R activation, SKF38393 (SKF), 100 μm; D₁R antagonism, SCH23390 (SCH), 100 μm] had no effect on the size of Cx36 plaques in AII amacrine cells. Inhibition of PKA [Rp-8-CPT-cAMPS (Rp), 20 μm] also had no effect on Cx36 plaque size. B, Conditions that altered Cx36 phosphorylation at Ser293 (D₁R activation, SKF38393, 100 μm; D₁R antagonism, SCH23390, 100 μm) had no effect on the overall distribution of Cx36 plaques in AII amacrine cells. Inhibition of PKA (Rp-8-CPT-cAMPS, 20 μm) also had no effect on Cx36 plaque distribution. Error bars are SEM, n = 5. No significant changes were found.

Figure 4.

PP2A is required for D₁R-dependent dephosphorylation of Cx36 at Ser293 in AII amacrine cells. A, B, Control; color scheme and antibodies are the same as Figure 2. C, D, D₁R activation [SKF38393 (SKF), 10 μm] greatly diminished Ser293-P labeling. E, F, Inhibition of PP2A [microcystin-LR (MC), 0.5 nm] completely blocked the reduction in Ser293 phosphorylation caused by D₁R activation. G, H, Inhibition of PP2A alone led to increased phosphorylation of Ser293. I, Summary of data shows that inhibition of PP2A significantly blocked the reduction in Ser293 phosphorylation caused by D₁R activation and that PP2A inhibition alone significantly increased Ser293 phosphorylation. J, Summary of data shows that changes in the percentage of Cx36 plaques that show detectable Ser293-P labeling follows the same pattern established for relative Ser293-P measurements in I. Error bars are SEM, n = 6. *p < 0.05, **p < 0.01. Images are 1-μm-deep stacks. Scale bar: H, 10 μm.

Figure 5.

PP1 negatively regulates the dephosphorylation of Cx36. A, B, Control; color scheme and antibodies are the same as Figure 2. C, D, D₁R activation [SKF38393 (SKF), 10 μm] greatly diminished Ser293-P labeling. E, F, Inhibition of PP1 [tautomycetin (TMC), 10 nm] did not alter the effects of D₁R activation. G, H, Inhibition of PP1 alone was sufficient to cause a strong reduction in Ser293-P labeling. I, Summary of data shows that inhibition of PP1 did not prevent the reduction in Ser293 phosphorylation caused by D₁R activation and that PP1 inhibition alone significantly reduced Ser293 phosphorylation. J, Summary of data shows that changes in the percentage of Cx36 plaques that show detectable Ser293-P labeling follows the same pattern established for relative Ser293-P measurements in I. Error bars are SEM, n = 4. *p < 0.05, **p < 0.01. Images are 1-μm-deep stacks. Scale bar: H, 10 μm.

Figure 6.

PP2A is required for PKA-dependent dephosphorylation of Cx36 at Ser293 in AII amacrine cells. A, B, Control; color scheme and antibodies are the same as Figure 2. C, D, PKA activation [Sp-8-CPT-cAMPS (Sp), 20 μm] greatly diminished Ser293-P labeling, just as D₁R activation did. E, F, Inhibition of PP2A [microcystin-LR (MC), 0.5 nm] completely blocked the reduction in Ser293 phosphorylation caused by PKA activation. G, H, Inhibition of PP2A alone slightly increased Ser293 phosphorylation relative to control. I, Summary of data shows that inhibition of PP2A significantly blocked the reduction in Ser293 phosphorylation caused by PKA activation. We observed a trend toward increased Ser293 phosphorylation when PP2A alone was inhibited, similar to the significant increase we observed previously with the same treatment (Fig. 4). J, Summary of data shows that changes in the percentage of Cx36 plaques that show detectable Ser293-P labeling follows the same pattern established for relative Ser293-P measurements in I. Error bars are SEM, n = 5. **p < 0.01. Images are 1-μm-deep stacks. Scale bar: H, 10 μm.

Figure 7.

Model of D₁R-dependent regulation of Cx36-mediated coupling between AII amacrine cells. Activation of D₁Rs (1) initiates a cascade leading to activation of PKA (2); both D₁R and PKA activation are sufficient to uncouple AII amacrine cells (Hampson et al., 1992; Mills and Massey, 1995). In this study, we showed that D₁R-dependent dephosphorylation (gray P) of Ser293 on Cx36 uncouples AII amacrine cells (3). We show that PP2A is required for both D₁R- and PKA-stimulated dephosphorylation of Ser293 (4). This provides evidence that the D₁R → PKA → PP2A pathway, which was recently described in spiny neurons in the striatum and led to dephosphorylation of Thr75 on DARPP-32 (Ahn et al., 2007), is also present in the AII amacrine cell. AII amacrine cells also express DARPP-32 (Partida et al., 2004; Witkovsky et al., 2007), and dephosphorylation of Thr75 on DARPP-32 facilitates PKA-mediated phosphorylation (black P) of Thr34 (5), which converts DAPRR-32 into an inhibitor of PP1 (6) (Svenningsson et al., 2004). We found that PP1 negatively regulates the dephosphorylation of Cx36, possibly by opposing PKA-mediated activation of PP2A (7). Our results indicate that the D₁R → PKA → PP2A pathway is not limited to striatal neurons and may represent a common pathway in neurons expressing D₁Rs and DARPP-32.