Protein kinase A-mediated phosphorylation of connexin36 in mouse retina results in decreased gap junctional communication between AII amacrine cells.
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.
Urschel
Stephanie
S
Institute of Genetics, University of Bonn, 53117 Bonn, Germany.
Höher
Thorsten
T
Schubert
Timm
T
Alev
Cantas
C
Söhl
Goran
G
Wörsdörfer
Philipp
P
Asahara
Takayuki
T
Dermietzel
Rolf
R
Weiler
Reto
R
Willecke
Klaus
K
D025042:Amacrine Cells / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002450:Cell Communication; D002460:Cell Line; D017630:Connexins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000242:Cyclic AMP / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D015536:Down-Regulation; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D017629:Gap Junctions / Q000378:metabolism*; D006801:Humans; D008027:Light; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D012160:Retina / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics; D014162:Transfection; D000097004:Gap Junction delta-2 Protein
D017630:Connexins; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D000097004:Gap Junction delta-2 Protein