Regulation of Gap Junction Coupling Through the Neuronal Connexin Cx35 by Nitric Oxide and cGMP

Patel, Leena; Mitchell, Cheryl K.; Dubinsky, William P.; O’Brien, John

doi:10.1080/15419060600631474

Cited by 51 publications

(51 citation statements)

References 49 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We conclude that Ser-110 and Ser-293 are the main phosphorylation sites in the Cx36 protein. This is in accordance to Patel et al (27), whose experiments with the fish ortholog Cx35 showed the same prevalence in the orthologous phosphorylation sites used. The PKA consensus motifs containing Ser-306 and Ser-315 seem to be used to a lesser extent.…”

Section: The Cytoplasmic Domains Of Cx36 Are Phosphorylated Insupporting

confidence: 92%

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

Urschel

Höher

Schubert

et al. 2006

Journal of Biological Chemistry

124

151

View full text Add to dashboard Cite

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...

show abstract

Section: The Cytoplasmic Domains Of Cx36 Are Phosphorylated Insupporting

confidence: 92%

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

Urschel

Höher

Schubert

et al. 2006

Journal of Biological Chemistry

124

151

View full text Add to dashboard Cite

show abstract

“…Second, the two identified regulatory phosphorylation sites appear to be targets for a number of signaling pathways in addition to the cAMP/PKA pathway. 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).…”

Section: Discussionmentioning

confidence: 73%

“…The nitric oxide/cGMP/PKG pathway has also been shown to affect Cx35 phosphorylation, and several other signaling pathways are predicted to (Patel et al, 2006). This raises the possibility that phosphorylation of the giant gap junctions is influenced by transmitters other than dopamine.…”

Section: Discussionmentioning

confidence: 99%

“…Uncoupling requires phosphorylation at two sites on Cx35, Ser110 in the intracellular loop and Ser276 in the carboxyl terminal tail. These same two sites are also the target of phosphorylation by cGMPdependent protein kinase (PKG) in vitro, suggesting that several signaling pathways may converge on these regulatory residues (Patel et al, 2006). We reasoned that the phosphorylation state of Ser110 and Ser276 should be indicative of the degree of coupling possible through Cx35 gap junctions.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

Kothmann

Li²,

Burr

et al. 2007

Vis Neurosci

Self Cite

View full text Add to dashboard Cite

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.

show abstract

“…45 However, it remains to be determined if there is any interaction between NO and connexins in the testes. Also, NO has a negative role in modulating integrin-linked kinase, a protein kinase related to the integrin-associated protein complex, in rat kidneys 46 (Table 2).…”

Section: Nos/no and Spermatogenesismentioning

confidence: 99%

Nitric Oxide and Cyclic Nucleotides: Their Roles in Junction Dynamics and Spermatogenesis

Lee

Cheng

2009

Advances in Experimental Medicine and Biology

View full text Add to dashboard Cite

Spermatogenesis is a highly complicated process in which functional spermatozoa (haploid, 1n) are generated from primitive mitotic spermatogonia (diploid, 2n). This process involves the differentiation and transformation of several types of germ cells as spermatocytes and spermatids undergo meiosis and differentiation. Due to its sophistication and complexity, testis possesses intrinsic mechanisms to modulate and regulate different stages of germ cell development under the intimate and indirect cooperation with Sertoli and Leydig cells, respectively. Furthermore, developing germ cells must translocate from the basal to the apical (adluminal) compartment of the seminiferous epithelium. Thus, extensive junction restructuring must occur to assist germ cell movement. Within the seminiferous tubules, three principal types of junctions are found namely anchoring junctions, tight junctions, and gap junctions. Other less studied junctions are desmosome-like junctions and hemidesmosome junctions. With these varieties of junction types, testes are using different regulators to monitor junction turnover. Among the uncountable junction modulators, nitric oxide (NO) is a prominent candidate due to its versatility and extensive downstream network. NO is synthesized by nitric oxide synthase (NOS). Three traditional NOS, specified as endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS), and one testis-specific nNOS (TnNOS) are found in the testis. For these, eNOS and iNOS were recently shown to have putative junction regulation properties. More important, these two NOSs likely rely on the downstream soluble guanylyl cyclase/cGMP/protein kinase G signaling pathway to regulate the structural components at the tight junctions and adherens junctions in the testes. Apart from the involvement in junction regulation, NOS/NO also participates in controlling the levels of cytokines and hormones in the testes. On the other hand, NO is playing a unique role in modulating germ cell viability and development, and indirectly acting on some aspects of male infertility and testicular pathological conditions. Thus, NOS/NO bears an irreplaceable role in maintaining the homeostasis of the microenvironment in the seminiferous epithelium via its different downstream signaling pathways.

show abstract

Regulation of Gap Junction Coupling Through the Neuronal Connexin Cx35 by Nitric Oxide and cGMP

Cited by 51 publications

References 49 publications

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

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

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

Nitric Oxide and Cyclic Nucleotides: Their Roles in Junction Dynamics and Spermatogenesis

Contact Info

Product

Resources

About