Electrical synapses can undergo activity-dependent plasticity. The calcium/calmodulin-dependent kinase II (CaMKII) appears to play a critical role in this phenomenon, but the underlying mechanisms of how CaMKII affects the neuronal gap junction protein connexin36 (Cx36) are unknown. Here we demonstrate effective binding of 35 S-labeled CaMKII to 2 juxtamembrane cytoplasmic domains of Cx36 and in vitro phosphorylation of this protein by the kinase. Both domains reveal striking similarities with segments of the regulatory subunit of CaMKII, which include the pseudosubstrate and pseudotarget sites of the kinase. Similar to the NR2B subunit of the NMDA receptor both Cx36 binding sites exhibit phosphorylation-dependent interaction and autonomous activation of CaMKII. CaMKII and Cx36 were shown to be significantly colocalized in the inferior olive, a brainstem nucleus highly enriched in electrical synapses, indicating physical proximity of these proteins. In analogy to the current notion of NR2B interaction with CaMKII, we propose a model that provides a mechanistic framework for CaMKII and Cx36 interaction at electrical synapses.brain ͉ electrical synapse ͉ gap junction ͉ protein-protein interaction ͉ synaptic plasticity

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...

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In the skin, multipotent keratinocyte stem cells (KSC) are localised in the hair follicle bulge region. Although, KSC can be cultivated and grown in two-dimensional (2D) culture they rapidly lose stem cell markers when isolated from their niche. Currently, there is no KSC culture method available which recapitulates an environment similar to the KSC niche in the hair follicle. Here we describe the successful establishment of an in vitro 3D stem cell culture model developed from clonally growing keratinocyte lines derived from neonatal mice using culture conditions previously established for human keratinocytes. After 20 passages, keratinocyte lines showed a stable ratio of holoclones (stem cells), meroclones (stem and precursor cells) and paraclones (differentiating cells), with approximately 29% holoclones, 54% meroclones and 17% paraclones, and were thus termed keratinocyte stem and precursor cell (KSPC) cultures. In high calcium medium, KSPC cultures grown at the air-liquid interphase differentiated and formed epidermal equivalents. Notably, and in contrast to primary keratinocytes, keratinocytes from KSPC cultures were able to aggregate and form spherical clusters in hanging drops, a characteristic hallmark shared with other stem cell types. Similar to the in vivo situation in the hair follicle bulge, KSPC aggregates also showed low proliferation, down-regulation of keratin 6, absence of keratin 1, and expression of the KSC markers keratin 15, Sox9, NFATc1 and Zfp145. KSPC aggregates therefore provide an optimal in vitro 3D environment for the further characterisation and study of normal and genetically modified KSPC.

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