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

Published online by Cambridge University Press:  20 July 2007

W. WADE KOTHMANN ,
XIAOFAN LI ,
GARY S. BURR  and
JOHN O'BRIEN
W. WADE KOTHMANN
Affiliation:
Department of Ophthalmology and Visual Science, University of Texas—Houston Medical School, Houston, Texas The Graduate School of Biomedical Sciences, University of Texas Health Science Center at Houston, Texas
XIAOFAN LI
Affiliation:
Department of Ophthalmology and Visual Science, University of Texas—Houston Medical School, Houston, Texas
GARY S. BURR
Affiliation:
Department of Ophthalmology and Visual Science, University of Texas—Houston Medical School, Houston, Texas Present address: Department of Wildlife and Fisheries Sciences, Texas A&M University, College Station, Texas
JOHN O'BRIEN
Affiliation:
Department of Ophthalmology and Visual Science, University of Texas—Houston Medical School, Houston, Texas The Graduate School of Biomedical Sciences, University of Texas Health Science Center at Houston, Texas
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Abstract

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 μm2) 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.

Keywords

Connexin36PhosphorylationLight adaptationPhospho-specific antibodiesGap junctions
Type
Research Article
Information
Visual Neuroscience , Volume 24 , Issue 3 , May 2007 , pp. 363 - 375
Copyright
© 2007 Cambridge University Press

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