Modulation of perch connexin35 hemi‐channels by cyclic AMP requires a protein kinase A phosphorylation site

Mitropoulou, Georgia; Bruzzone, Roberto

doi:10.1002/jnr.10572

Cited by 48 publications

(48 citation statements)

References 85 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Mitropoulou & Bruzzone (2003) found that a consensus PKA recognition sequence in the intracellular loop of perch Cx35 was essential for the 8-Br-cAMP-induced reduction in hemichannel currents observed in Xenopus oocytes. They further found that recreating this recognition sequence in skate Cx35, which was unresponsive to 8-Br-cAMP in its wild-type form in Xenopus oocytes, conferred the same behavior to this connexin.…”

Section: Discussionmentioning

confidence: 95%

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

Kothmann

Li²,

Burr

et al. 2007

Vis Neurosci

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

Section: Discussionmentioning

confidence: 95%

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

Kothmann

Li²,

Burr

et al. 2007

Vis Neurosci

View full text Add to dashboard Cite

show abstract

“…Mitropoulou and Bruzzone [30] recently reported that perch Cx35 hemichannels were regulated by cAMP analogs in Xenopus oocytes. They found that conversion of the PKA recognition site associated with the Ser110 phosphorylation site to the sequence found in skate Cx35 eliminated regulation by cAMP analogs.…”

Section: Discussionmentioning

confidence: 99%

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

Ouyang

Winbow

Patel

et al. 2005

Molecular Brain Research

View full text Add to dashboard Cite

Connexin 35 (Cx35) is a major component of electrical synapses in the central nervous system. Many gap junctions containing Cx35 are regulated by dopamine receptor pathways that involve protein kinase A (PKA). To study the mechanism of PKA regulation, we analyzed direct phosphorylation of Cx35 by PKA in vitro, and studied the regulation of Neurobiotin tracer coupling in HeLa cells expressing Cx35 or Cx35 mutants that lack phosphorylation sites. In Cx35-transfected cells, application of the PKA activator Sp-8-cpt-cAMPS caused a significant decline in coupling, while a PKA inhibitor, Rp-8-cpt-cAMPS, significantly increased tracer coupling. In vitro phosphorylation and mutagenic analysis showed that PKA phosphorylates Cx35 directly at two major sites, Ser110 in the intracellular loop and Ser276 in the carboxyl terminus. In addition, a minor phosphorylation site in the C-terminus was identified by truncation of the last 7 amino acids at Ser298. The mutations Ser110Ala or Ser276Ala significantly reduced regulation of coupling by the PKA activator, while a combination of the two eliminated regulation. Truncation at Ser298 reversed the regulation such that the PKA activator significantly increased and the PKA inhibitor significantly decreased coupling. The activation was eliminated in the S110A,S276A,S298ter triple mutant. We conclude that PKA regulates Cx35 coupling in a complex manner that requires both major phosphorylation sites. Furthermore, the tip of the C-terminus acts as a "switch" that determines whether phosphorylation will inhibit or enhance coupling. Reliance on the combined states of three sites provides fine control over the degree of coupling through Cx35 gap junctions.

show abstract

“…(Opening was detected by dye uptake, which was prevented by gap-junction blockers.) Hemichannels in teleost and elasmobranch horizontal cells are opened by quinine and by depolarization in low-Ca 2+ solution; dopamine-receptor and cAMP agonists reduce hemichannel and cell-cell channel conductance in these systems [34][35][36][37][38]. The connexins in these cases are paralogs of the (largely) neuron-specific mammalian connexin Cx36.…”

Section: What Opens Hemichannels?mentioning

confidence: 99%

New roles for astrocytes: Gap junction hemichannels have something to communicate

Bennett

Contreras

Bukauskas

et al. 2003

Trends in Neurosciences

371

279

View full text Add to dashboard Cite

Gap junctions are clusters of aqueous channels that connect the cytoplasm of adjoining cells. Each cell contributes a hemichannel, or connexon, to each cell-cell channel. The cell-cell channels are permeable to relatively large molecules, and it was thought that opening of hemichannels to the extracellular space would kill cells through loss of metabolites, collapse of ionic gradients and influx of Ca 2+ . Recent findings indicate that specific non-junctional hemichannels do open under both physiological and pathological conditions, and that opening is functional or deleterious depending on the situation. Most of these studies utilized cells in tissue culture that expressed a specific gap junction protein, connexin 43. Several such examples are reviewed here, with a particular focus on astrocytes.Gap junctions mediate intercellular communication by providing ultrastructural cytoplasmic continuity, and they are integral to formation of the functional syncytium of astrocytes. Each of the joined cells contributes a hemichannel or connexon to each cell-cell channel ( Figure 1; Box 1). Each hemichannel comprises a hexamer of connexins arranged around a central pore, and the cell-cell channels are gated by several stimuli, including transjunctional voltage, low pH and various pharmacological agents. Connexins are encoded by a gene family with at least 20 members in mammals [1].Gap-junction pores are nominally 1.0-1.5 nm in diameter and are permeable to molecules of 1 kDa [2]. Junctions formed from connexin 43 (Cx43), the predominant connexin in astrocytes, are permeable to Lucifer Yellow (443 Da, −2 charge) and propidium (420 Da, +2 charge). Other connexins appear to be more charge selective: Cx32 is more permeable to anions and Cx45 is more permeable to cations [2]. Single-channel conductance varies widely, from ~15 pS for Cx36 [3] and 110 pS for Cx43 [4] to ~375 pS for Cx37 [5]. The maximum size of permeant species also varies. Such diversity in selectivity and conductance are likely to account for the expansion of the connexin family. Why shouldn't hemichannels be open?Given the permeability and conductance of gap junctions, and the expectation that an open hemichannel would have twice the conductance and permeability of a cell-cell channel (in terms of ion or molecular flux, rather than selectivity), it was thought unlikely that hemichannels in the non-junctional membrane would open [6]. Direct evidence for this was provided by measurements during formation of the first channel between a newly apposed pair of cells. Each cell was held at a different potential, and then when the first channel NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript opened, increased current was seen in the cell held at a more positive potential and decreased (more negative) current was seen in the other cell, but the sum of the currents clamping the two cells remained constant [7]. Thus, there was no change in the current flowing into the extracellular space, and the hemichannels were closed before openin...

show abstract

Modulation of perch connexin35 hemi‐channels by cyclic AMP requires a protein kinase A phosphorylation site

Cited by 48 publications

References 85 publications

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

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

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

New roles for astrocytes: Gap junction hemichannels have something to communicate

Contact Info

Product

Resources

About