Protein kinase A mediates regulation of gap junctions containing connexin35 through a complex pathway

Ouyang, Xiaosen; Winbow, V.M.; Patel, Leena; Burr, Gary S.; Mitchell, Cheryl K.; O’Brien, John

doi:10.1016/j.molbrainres.2004.10.045

Cited by 53 publications

(97 citation statements)

References 52 publications

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“…A preponderance of evidence from retinal neurons suggests that gap junctional coupling through Cx35/36 can be regulated by environmental conditions such as light exposure and by neurotransmitter signaling pathways. Comparable regulation has been observed directly for Cx35 in cell culture expression systems and has been shown to depend on direct phosphorylation of the connexin (Ouyang et al, 2005;Patel et al, 2006). The outcomes of these studies indicate that two phosphorylation sites are critical for regulation of coupling by the cAMP/PKA pathway, and perhaps the cGMP/PKG pathway as well.…”

Section: Cx35/36 Is Regulated By Phosphorylation At Conserved Sitesmentioning

confidence: 67%

“…Phosphorylated synthetic peptides corresponding to the two major regulatory PKA phosphorylation sites of perch Cx35 (Ouyang et al, 2005) were made at the M.D. Anderson Cancer Center Synthetic Antigen Laboratory.…”

Section: Development Of Phospho-specific Antibodiesmentioning

confidence: 99%

“…GST fusion proteins containing the cytoplasmic intracellular loop and carboxyl terminal domains of perch Cx35 Ouyang et al, 2005) were expressed in E. coli strain BL21 and purified by binding to glutathione sepharose 4B (Amersham, Piscataway, NJ). Fusion proteins (0.5 to 1 μg protein per reaction) were incubated with 0.5 units cAMPdependent protein kinase (PKA) catalytic subunit (mouse α isoform; New England Biolabs, Beverly, MA) for 90 min.…”

Section: In Vitro Phosphorylationmentioning

confidence: 99%

“…The outcomes of these studies indicate that two phosphorylation sites are critical for regulation of coupling by the cAMP/PKA pathway, and perhaps the cGMP/PKG pathway as well. Figure 1A shows these two sites, Ser110 in the intracellular loop and Ser276 in the C-terminal domain (Ser276 in teleost, amphibian, and avian Cx35 is equivalent to Ser293 of mammalian Cx36 and Ser273 of skate Cx35), and a putative model of regulation by PKA (adapted from Ouyang et al, 2005). In this model, phosphorylation at both Ser110 and Ser276 is required to cause substantial uncoupling of Cx35 gap junctions; phosphorylation of either residue alone causes only very modest uncoupling (Ouyang et al, 2005).…”

Section: Cx35/36 Is Regulated By Phosphorylation At Conserved Sitesmentioning

confidence: 99%

“…The effect of PKA activity on coupling in AII amacrine cells has been duplicated with Cx35 stably expressed in a mammalian cell line (Ouyang et al, 2005). Activation of PKA causes uncoupling while PKA inhibition causes an increase in coupling.…”

Section: Introductionmentioning

confidence: 99%

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

Kothmann

Li²,

Burr

et al. 2007

Vis Neurosci

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Connexin 35/36 is the most widespread neuronal gap junction protein in the retina and central nervous system. Electrical and/or tracer coupling in a number of neuronal circuits that express this connexin are regulated by light adaptation. In many cases the regulation of coupling depends on signaling pathways that activate protein kinases such as PKA, and Cx35 has been shown to be regulated by PKA phosphorylation in cell culture systems. To examine whether phosphorylation might regulate Cx35/36 in the retina we developed phospho-specific polyclonal antibodies against the two regulatory phosphorylation sites of Cx35 and examined the phosphorylation state of this connexin in the retina. Western blot analysis with hybrid bass retinal membrane preparations showed Cx35 to be phosphorylated at both the Ser110 and Ser276 sites, and this labeling was eliminated by alkaline phosphatase digestion. The homologous sites of mouse and rabbit Cx36 were also phosphorylated in retinal membrane preparations. Quantitative confocal immunofluorescence analysis showed gap junctions identified with a monoclonal anti-Cx35 antibody to have variable levels of phosphorylation at both the Ser110 and Ser276 sites. Unusual gap junctions that could be identified by their large size (up to 32 μm 2 ) and location in the IPL showed a prominent shift in phosphorylation state from heavily phosphorylated in nighttime, dark-adapted retina to weakly phosphorylated in daytime, light-adapted retina. Both Ser110 and Ser276 sites showed significant changes in this manner. Under both lighting conditions other gap junctions varied from non-phosphorylated to heavily phosphorylated. We predict that changes in the phosphorylation states of these sites correlate with changes in the degree of coupling through Cx35/36 gap junctions. This leads to the conclusion that connexin phosphorylation mediates changes in coupling in some retinal networks. However, these changes are not global and likely occur in a cell type-specific or possibly a gap junction-specific manner.

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Section: Cx35/36 Is Regulated By Phosphorylation At Conserved Sitesmentioning

confidence: 67%

Section: Development Of Phospho-specific Antibodiesmentioning

confidence: 99%

Section: In Vitro Phosphorylationmentioning

confidence: 99%

Section: Cx35/36 Is Regulated By Phosphorylation At Conserved Sitesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Kothmann

Li²,

Burr

et al. 2007

Vis Neurosci

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Zebrafish connexin 79.8 (Gja8a): A lens connexin used as an electrical synapse in some neurons

Yoshikawa

Vila

Segelken

et al. 2016

Developmental Neurobiology

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In the mammalian central nervous system a remarkably small number of connexins is used in electrical synapses, with the majority formed from Cx36. A larger number has been detected in teleosts, with some seeming to serve restricted roles. Here we report the discovery of a new connexin expressed in the zebrafish lens and a limited set of neurons. Zebrafish cx79.8 (gja8a), previously annotated incorrectly as cx50.5 based on a partial cDNA sequence, is a homologue of mammalian Cx50 (Gja8). We examined its expression through transgenic promoter-reporter constructs, in situ hybridization, and immunolabeling, and examined regulation of coupling in transfected HeLa cells. cx79.8 was expressed most strongly in the lens, but expression was also found in several groups of neurons in the cerebellum and related areas at the midbrain-hindbrain boundary, in cone photoreceptors, and in neurons in the retinal inner nuclear and ganglion cell layers. Labeling in the retina with antibodies against two C-terminal epitopes revealed numerous small punctate spots in the inner plexiform layer and along the somata of cones. Abundant gap junctions were labeled in the outer 1/3 of the lens, but were absent from the center, suggesting that the epitopes or the entire protein was absent from the center. Cx79.8 tracer coupling was strongly regulated by phosphorylation, and was extremely low in control conditions in HeLa cells due to protein phosphatase 2A activity. These properties allow coupling to be strongly restricted in situ, a frequently observed property for electrical synapses.

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Neuronal coupling via connexin36 contributes to spontaneous synaptic currents of striatal medium‐sized spiny neurons

Cummings

Yamazaki

Cepeda

et al. 2008

J of Neuroscience Research

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Gap junctions provide a means for electrotonic coupling between neurons, allowing for the generation of synchronous activity, an important contributor to learning and memory. Connexin36 (Cx36) is largely neuron specific and provides a target for genetic manipulation to determine the physiological relevance of neuronal coupling. Within the striatum, Cx36 is more specifically localized to the interneuronal population, which provides the main inhibitory input to the principal projection medium-sized spiny neurons. In the present study, we examined the impact of genetic ablation of Cx36 on striatal spontaneous synaptic activity. Patch-clamp recordings were performed from medium-sized spiny neurons, the primary target of interneurons. In Cx36 knockout mice, the frequencies of both excitatory and inhibitory spontaneous postsynaptic currents were reduced. We also showed that activation of dopamine receptors differentially modulated the frequency of GABAergic currents in Cx36 knockout mice compared with their wild-type littermates, suggesting that dopamine plays a role in altering the coupling of interneurons. Taken together, the present findings demonstrate that electrical coupling of neuronal populations is important for the maintenance of normal chemical synaptic interactions within the striatum.

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Protein kinase A mediates regulation of gap junctions containing connexin35 through a complex pathway

Cited by 53 publications

References 52 publications

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

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

Zebrafish connexin 79.8 (Gja8a): A lens connexin used as an electrical synapse in some neurons

Neuronal coupling via connexin36 contributes to spontaneous synaptic currents of striatal medium‐sized spiny neurons

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