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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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References

REFERENCES

Attwell, D., Borges, S., Wu, S.M. & Wilson, M. (1987). Signal clipping by the rod output synapse. Nature 328, 522–524.CrossRefGoogle Scholar
Baldridge, W.H. & Ball, A.K. (1991). Background illumination reduces horizontal cell receptive-field size in both normal and 6-hydroxydopamine-lesioned goldfish retinas. Visual Neuroscience 7, 441–450.CrossRefGoogle Scholar
Bloomfield, S.A. & Volgyi, B. (2004). Function and plasticity of homologous coupling between AII amacrine cells. Vision Research 44, 3297–3306.CrossRefGoogle Scholar
Bloomfield, S.A. & Xin, D. (1997). A comparison of receptive-field and tracer-coupling size of amacrine and ganglion cells in the rabbit retina. Visual Neuroscience 14, 1153–1165.CrossRefGoogle Scholar
Bloomfield, S.A., Xin, D. & Osborne, T. (1997). Light-induced modulation of coupling between AII amacrine cells in the rabbit retina. Visual Neuroscience 14, 565–576.CrossRefGoogle Scholar
Bloomfield, S.A., Xin, D. & Persky, S.E. (1995). A comparison of receptive field and tracer coupling size of horizontal cells in the rabbit retina. Visual Neuroscience 12, 985–999.CrossRefGoogle Scholar
Burr, G.S., Mitchell, C.K., Keflemariam, Y.J., Heidelberger, R. & O'Brien, J. (2005). Calcium-dependent binding of calmodulin to neuronal gap junction proteins. Biochemical and Biophysical Research Communications 335, 1191–1198.CrossRefGoogle Scholar
Cohen, A.I., Todd, R.D., Harmon, S. & O'Malley, K.L. (1992). Photoreceptors of mouse retinas possess D4 receptors coupled to adenylate cyclase. Proceedings of the National Academy of Sciences USA 89, 12093–12097.CrossRefGoogle Scholar
Condorelli, D.F., Parenti, R., Spinella, F., Salinaro, A.T., Belluardo, N., Cardile, V. & Cicirata, F. (1998). Cloning of a new gap junction gene (Cx36) highly expressed in mammalian brain neurons. European Journal of Neuroscience 10, 1202–1208.CrossRefGoogle Scholar
Cook, J.E. & Becker, D.L. (1995). Gap junctions in the vertebrate retina. Microscopy Research & Technique 31, 408–419.CrossRefGoogle Scholar
Copenhagen, D.R. & Green, D.G. (1987). Spatial spread of adaptation within the cone network of turtle retina. Journal of Physiology (London) 393, 763–776.CrossRefGoogle Scholar
Deans, M.R., Gibson, J.R., Sellitto, C., Connors, B.W. & Paul, D.L. (2001). Synchronous activity of inhibitory networks in neocortex requires electrical synapses containing connexin36. Neuron 31, 477–485.CrossRefGoogle Scholar
Deans, M.R., Volgyi, B., Goodenough, D.A., Bloomfield, S.A. & Paul, D.L. (2002). Connexin36 is essential for transmission of rod-mediated visual signals in the mammalian retina. Neuron 36, 703–712.CrossRefGoogle Scholar
Dearry, A. & Burnside, B. (1986). Dopaminergic regulation of cone retinomotor movement in isolated teleost retinas: I. Induction of cone contraction is mediated by D2 receptors. Journal of Neurochemistry 46, 1006–1021.Google Scholar
DeVries, S.H. & Schwartz, E.A. (1989). Modulation of an electrical synapse between solitary pairs of catfish horizontal cells by dopamine and second messengers. Journal of Physiology (London) 414, 351–375.CrossRefGoogle Scholar
Feigenspan, A., Janssen-Bienhold, U., Hormuzdi, S., Monyer, H., Degen, J., Sohl, G., Willecke, K., Ammermuller, J. & Weiler, R. (2004). Expression of connexin36 in cone pedicles and OFF-cone bipolar cells of the mouse retina. Journal of Neuroscience 24, 3325–3334.Google Scholar
Feigenspan, A., Teubner, B., Willecke, K. & Weiler, R. (2001). Expression of neuronal connexin36 in AII amacrine cells of the mammalian retina. Journal of Neuroscience 21, 230–239.Google Scholar
Guldenagel, M., Ammermuller, J., Feigenspan, A., Teubner, B., Degen, J., Sohl, G., Willecke, K. & Weiler, R. (2001). Visual transmission deficits in mice with targeted disruption of the gap junction gene connexin36. Journal of Neuroscience 21, 6036–6044.Google Scholar
Hampson, E.C., Vaney, D.I. & Weiler, R. (1992). Dopaminergic modulation of gap junction permeability between amacrine cells in mammalian retina. Journal of Neuroscience 12, 4911–4922.Google Scholar
Harsanyi, K. & Mangel, S.C. (1992). Activation of a D2 receptor increases electrical coupling between retinal horizontal cells by inhibiting dopamine release. Proceedings of the National Academy of Sciences USA 89, 9220–9224.CrossRefGoogle Scholar
Hidaka, S., Kato, T. & Hashimoto, Y. (2005). Structural and functional properties of homologous electrical synapses between retinal amacrine cells. Journal of Integrative Neuroscience 4, 313–340.CrossRefGoogle Scholar
Hidaka, S., Maehara, M., Umino, O., Lu, Y. & Hashimoto, Y. (1993). Lateral gap junction connections between retinal amacrine cells summating sustained responses. Neuroreport 5, 29–32.CrossRefGoogle Scholar
Hormuzdi, S.G., Pais, I., Lebeau, F.E., Towers, S.K., Rozov, A., Buhl, E.H., Whittington, M.A. & Monyer, H. (2001). Impaired electrical signaling disrupts gamma frequency oscillations in connexin 36-deficient mice. Neuron 31, 487–495.CrossRefGoogle Scholar
Iuvone, P.M., Galli, C.L., Garrison-Gund, C.K. & Neff, N.H. (1978). Light stimulates tyrosine hydroxylase activity and dopamine synthesis in retinal amacrine neurons. Science 202, 901–902.CrossRefGoogle Scholar
Kramer, S.G. (1971). Dopamine: A retinal neurotransmitter. I. Retinal uptake, storage, and light-stimulated release of H3-dopamine in vivo. Investigative Ophthalmology 10, 438–452.Google Scholar
Krizaj, D., Gabriel, R., Owen, W.G. & Witkovsky, P. (1998). Dopamine D2 receptor-mediated modulation of rod-cone coupling in the Xenopus retina. Journal of Comparative Neurology 398, 529–538.3.0.CO;2-4>CrossRefGoogle Scholar
Kwak, B.R., Hermans, M.M., De Jonge, H.R., Lohmann, S.M., Jongsma, H.J. & Chanson, M. (1995). Differential regulation of distinct types of gap junction channels by similar phosphorylating conditions. Molecular Biology of the Cell 6, 1707–1719.CrossRefGoogle Scholar
Lamb, T.D. & Simon, E.J. (1976). The relation between intercellular coupling and electrical noise in turtle photoreceptors. Journal of Physiology (London) 263, 257–286.CrossRefGoogle Scholar
Lampe, P.D. (1994). Analyzing phorbol ester effects on gap junctional communication: A dramatic inhibition of assembly. Journal of Cell Biology 127, 1895–1905.CrossRefGoogle Scholar
Lampe, P.D., Tenbroek, E.M., Burt, J.M., Kurata, W.E., Johnson, R.G. & Lau, A.F. (2000). Phosphorylation of connexin43 on serine368 by protein kinase C regulates gap junctional communication. Journal of Cell Biology 149, 1503–1512.CrossRefGoogle Scholar
Lankheet, M.J., Przybyszewski, A.W. & Van De Grind, W.A. (1993). The lateral spread of light adaptation in cat horizontal cell responses. Vision Research 33, 1173–1184.CrossRefGoogle Scholar
Lasater, E.M. (1987). Retinal horizontal cell gap junctional conductance is modulated by dopamine through a cyclic AMP-dependent protein kinase. Proceedings of the National Academy of Sciences USA 84, 7319–7323.CrossRefGoogle Scholar
Maier, N., Guldenagel, M., Sohl, G., Siegmund, H., Willecke, K. & Draguhn, A. (2002). Reduction of high-frequency network oscillations (ripples) and pathological network discharges in hippocampal slices from connexin 36-deficient mice. Journal of Physiology 541, 521–528.CrossRefGoogle Scholar
Marc, R.E., Liu, W.L. & Muller, J.F. (1988). Gap junctions in the inner plexiform layer of the goldfish retina. Vision Research 28, 9–24.CrossRefGoogle Scholar
Mills, S.L., O'Brien, J.J., Li, W., O'Brien, J. & Massey, S.C. (2001). Rod pathways in the mammalian retina use connexin36. Journal of Comparative Neurology 436, 336–350.CrossRefGoogle Scholar
Mitropoulou, G. & Bruzzone, R. (2003). Modulation of perch connexin35 hemi-channels by cyclic AMP requires a protein kinase A phosphorylation site. Journal of Neuroscience Research 72, 147–157.CrossRefGoogle Scholar
Moreno, A.P., Saez, J.C., Fishman, G.I. & Spray, D.C. (1994). Human connexin43 gap junction channels. Regulation of unitary conductances by phosphorylation. Circulation Research 74, 1050–1057.Google Scholar
Nir, I., Harrison, J.M., Haque, R., Low, M.J., Grandy, D.K., Rubinstein, M. & Iuvone, P.M. (2002). Dysfunctional light-evoked regulation of cAMP in photoreceptors and abnormal retinal adaptation in mice lacking dopamine D4 receptors. Journal of Neuroscience 22, 2063–2073.Google Scholar
O'Brien, J., Al-Ubaidi, M.R. & Ripps, H. (1996). Connexin 35: A gap-junctional protein expressed preferentially in the skate retina. Molecular Biology of the Cell 7, 233–243.CrossRefGoogle Scholar
O'Brien, J., Bruzzone, R., White, T.W., Al-Ubaidi, M.R. & Ripps, H. (1998). Cloning and expression of two related connexins from the perch retina define a distinct subgroup of the connexin family. Journal of Neuroscience 18, 7625–7637.Google Scholar
O'Brien, J., Nguyen, H.B. & Mills, S.L. (2004). Cone photoreceptors in bass retina use two connexins to mediate electrical coupling. Journal of Neuroscience 24, 5632–5642.Google Scholar
Ouyang, X., Winbow, V.M., Patel, L.S., Burr, G.S., Mitchell, C.K. & O'Brien, J. (2005). Protein kinase A mediates regulation of gap junctions containing connexin35 through a complex pathway. Brain Research, Molecular Brain Research 135, 1–11.Google Scholar
Pais, I., Hormuzdi, S.G., Monyer, H., Traub, R.D., Wood, I.C., Buhl, E.H., Whittington, M.A. & Lebeau, F.E. (2002). Sharp wave-like activity in the hippocampus in vitro in mice lacking the gap junction protein connexin 36. Journal of Neurophysiology 89, 2046–2054.CrossRefGoogle Scholar
Patel, L.S., Mitchell, C.K., Dubinsky, W.P. & O'Brien, J. (2006). Regulation of gap junction coupling through the neuronal connexin Cx35 by nitric oxide and cGMP. Cell Communication and Adhesion 13, 41–54.CrossRefGoogle Scholar
Pereda, A., O'Brien, J., Nagy, J.I., Bukauskas, F., Davidson, K.G., Kamasawa, N., Yasumura, T. & Rash, J.E. (2003). Connexin35 mediates electrical transmission at mixed synapses on Mauthner cells. Journal of Neuroscience 23, 7489–7503.Google Scholar
Schwartz, E.A. (1975a). Cones excite rods in the retina of the turtle. Journal of Physiology 246, 639–651.Google Scholar
Schwartz, E.A. (1975b). Rod-rod interaction in the retina of the turtle. Journal of Physiology 246, 617–638.Google Scholar
Sitaramayya, A., Crabb, J.W., Matesic, D.F., Margulis, A., Singh, V., Pulukuri, S. & Dang, L. (2003). Connexin 36 in bovine retina: Lack of phosphorylation but evidence for association with phosphorylated proteins. Visual Neuroscience 20, 385–395.CrossRefGoogle Scholar
Smith, R.G., Freed, M.A. & Sterling, P. (1986). Microcircuitry of the dark-adapted cat retina: functional architecture of the rod-cone network. Journal of Neuroscience 6, 3505–3517.Google Scholar
Sohl, G., Degen, J., Teubner, B. & Willecke, K. (1998). The murine gap junction gene connexin36 is highly expressed in mouse retina and regulated during brain development. FEBS Letters 428, 27–31.CrossRefGoogle Scholar
Teranishi, T. & Negishi, K. (1994). Double-staining of horizontal and amacrine cells by intracellular injection with lucifer yellow and biocytin in carp retina. Neuroscience 59, 217–226.CrossRefGoogle Scholar
Teranishi, T., Negishi, K. & Kato, S. (1984). Dye coupling between amacrine cells in carp retina. Neuroscience Letters 51, 73–78.CrossRefGoogle Scholar
Thompson, J.D., Higgins, D.G. & Gibson, T.J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research 22, 4673–4680.CrossRefGoogle Scholar
Tsang, V.C. & Wilkins, P.P. (1991). Optimum dissociating condition for immunoaffinity and preferential isolation of antibodies with high specific activity. Journal of Immunological Methods 138, 291–299.CrossRefGoogle Scholar
Urschel, S., Hoher, T., Schubert, T., Alev, C., Sohl, G., Worsdorfer, P., Asahara, T., Dermietzel, R., Weiler, R. & Willecke, K. (2006). Protein kinase A mediated phosphorylation of connexin36 in mouse retina results in decreased gap junctional communication between AII amacrine cells. Journal of Biological Chemistry 281, 33163–33171.CrossRefGoogle Scholar
Van Veen, T.A., Van Rijen, H.V. & Jongsma, H.J. (2000). Electrical conductance of mouse connexin45 gap junction channels is modulated by phosphorylation. Cardiovascular Research 46, 496–510.CrossRefGoogle Scholar
Wu, S.M. & Yang, X.L. (1988). Electrical coupling between rods and cones in the tiger salamander retina. Proceedings of the National Academy of Sciences USA 85, 275–278.Google Scholar
Yang, X.L. & Wu, S.M. (1989). Modulation of rod-cone coupling by light. Science 244, 352–354.Google Scholar
Zampighi, G.A., Planells, A.M., Lin, D. & Takemoto, D. (2005). Regulation of lens cell-to-cell communication by activation of PKCγ and disassembly of Cx50 channels. Investigative Ophthalmology and Visual Science 46, 3247–3255.Google Scholar
Zhang, J. & Wu, S.M. (2004). Connexin35/36 gap junction proteins are expressed in photoreceptors of the tiger salamander retina. Journal of Comparative Neurology 470, 1–12.Google Scholar
Zoidl, G., Meier, C., Petrasch-Parwez, E., Zoidl, C., Habbes, H.W., Kremer, M., Srinivas, M., Spray, D.C. & Dermietzel, R. (2002). Evidence for a role of the N-terminal domain in subcellular localization of the neuronal connexin36 (Cx36). Journal of Neuroscience Research 69, 448–465.Google Scholar