Protein Kinase A-mediated Phosphorylation of Connexin36 in Mouse Retina Results in Decreased Gap Junctional Communication between AII Amacrine Cells

Abstract

Gap junctions in AII amacrine cells of mammalian retina participate in the coordination of the rod and cone signaling pathway involved in visual adaptation. Upon stimulation by light, released dopamine binds to D 1 receptors on AII amacrine cells leading to increased intracellular cAMP (cyclic adenosine monophosphate) levels. AII amacrine cells express the gap junctional protein connexin36 (Cx36). Phosphorylation of Cx36 has been hypothesized to regulate gap junctional activity of AII amacrine cells. However, until now in vivo phosphorylation of Cx36 has not been reported. Indeed, it had been concluded that Cx36 in bovine retina is not phosphorylated, but in vitro phosphorylation for Cx35, the bass ortholog of Cx36, had been shown. To clarify this experimental discrepancy, we examined protein kinase A (PKA)-induced phosphorylation of Cx36 in mouse retina as a possible mechanism to modulate the extent of gap junctional coupling. The cytoplasmic domains of Cx36 and the total Cx36 protein were phosphorylated in vitro by PKA. Mass spectroscopy revealed that all four possible PKA consensus motifs were phosphorylated; however, domains point mutated at the sites in question showed a prevalent usage of Ser-110 and Ser-293. Additionally, we demonstrated that Cx36 was phosphorylated in cultured mouse retina. Furthermore, activation of PKA increased the level of phosphorylation of Cx36. cAMP-stimulated, PKA-mediated phosphorylation of Cx36 protein was accompanied by a decrease of tracer coupling between AII amacrine cells. Our results link increased phosphorylation of Cx36 to down-regulation of permeability through gap junction channels mediating light adaptation in the retina.The mammalian retina is a structure with three neuronal layers and two synaptic layers in which retinal neurons are not only forming numerous chemical synaptic contacts but also electrically and chemically coupled networks via gap junction channels. Gap junctions were reported in rod and cone photoreceptor cells, cone bipolar cells, horizontal cells, various subtypes of amacrine cells, and ganglion cells (1). Interneuronal communication through gap junction channels is essential for the rod pathway under conditions of scotopic illumination. Visual information from the rods is carried via rod bipolar cells to AII amacrine cells that form heterologous electrical synapses with ON cone bipolar cells, thus transferring rod signals to ganglion cells (2-4). Junctional hemichannels on the AII amacrine cell side consist of Cx36, 2 whereas Cx36 and/or Cx45 protein form electrical synapses on the bipolar cell side (5-7). Additionally, AII amacrine cells form homotypic gap junctions to neighboring AII amacrine cells, involving Cx36 (2, 3, 8 -10). Under scotopic conditions, the AII network only shows weak coupling and thus supports optimal transfer of the rod signal to the ON cone bipolar cells. Under mesopic light conditions, AII amacrine cells form an extensively coupled network. Pooling visual signals under this light condition increases sensitivity of AII ama...

Connexin 35/36 is phosphorylated at regulatory sites in the retina

“…We have found that PKG also phosphorylates both of these sites in vitro (Patel et al, 2006). These sites are also consensus phosphorylation sequences for Casein Kinase II, Ca 2+ -Calmodulin-dependent kinases, and S6 Kinase II (summarized in figure 1; see also Sohl et al, 1998;Urschel et al, 2006). Not all of these kinases are predicted to phosphorylate both sites, which emphasizes that the sites are not equivalent.…”

“…PKA phosphorylation at the homologous sites has been confirmed for mouse Cx36 and parallel regulation by PKA phosphorylation has been proposed (Urschel et al, 2006), although direct regulation by phosphorylation was not tested. Nonetheless, the high degree of sequence homology between these connexin gene homologues suggests that the mammalian Cx36 is regulated in the same fashion as fish Cx35.…”

Electrical synapses between AII amacrine cells in the retina: Function and modulation

“…Molecular studies suggest that Cx36 can be phosphorylated both by CaMKII and PKA (Alev et al, 2008;Urschel et al, 2006), strengthening the potential similarity with goldfish electrical synapses and their modulatory control (Cachope et al, 2007). On this background, it seems pertinent to review the potential sources of Ca 2+ influx in AII amacrine cells (Fig.…”

“…The first pharmacological evidence was obtained for activation of D1-type dopamine receptors in rabbit retina (Hampson et al, 1992;Mills and Massey, 1995;Xia and Mills, 2004) and has since been verified for mouse retina (Urschel et al, 2006). The strength or extent of coupling in these studies is measured by intracellular injection of tracer into a single cell, waiting a fixed time period, and then counting the number of cells to which the tracer diffused during a fixed time period.…”

“…While a later study by Mills and Massey (1995) confirmed the basic observations of Hampson et al (1992), neither study provided any direct evidence for a mechanism of modulatory control. Urschel et al (2006) found evidence that Cx36 could be phosphorylated by PKA at specific sites and suggested that this would correspond to decreased gap junction conductance.…”