Phosphorylation of connexin 50 by protein kinase A enhances gap junction and hemichannel function

Jialu Liu, Jose F Ek Vitorin, Susan T. Weintraub, Sumin Gu, Qian Shi, Janis M Burt, Jean X. Jiang

Research output: Contribution to journalArticle

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Abstract

Phosphorylation of connexins is an important mechanism regulating gap junction channels. However, the role(s) of connexin (Cx) phosphorylation in vivo are largely unknown. Here, we showed by mass spectrometry that Ser-395 in the C terminus of chicken Cx50 was phosphorylated in the lens. Ser-395 is located within a PKA consensus site. Analyses of Cx50 phosphorylation by two-dimensional thin layer chromatography tryptic phosphopeptide profiles suggested that Ser-395 was targeted by PKA in vivo. PKA activation increased both gap junction dye coupling and hemichannel dye uptake in a manner not involving increases in total Cx50 expression or relocation to the cell surface or gap junctional plaques. Single channel recordings indicated PKA enhanced transitions between the closed and ∼200-pS open state while simultaneously reducing transitions between this open state and a ∼65-pS subconductance state. The mutation of Ser-395 to alanine significantly attenuated PKA-induced increases in dye coupling and uptake by Cx50. However, channel records indicated that phosphorylation at this site was unnecessary for enhanced transitions between the closed and ∼200-pS conductance state. Together, these results suggest that Cx50 is phosphorylated in vivo by PKA at Ser-395 and that this event, although unnecessary for PKA-induced alterations in channel conductance, promotes increased dye permeability of Cx50 channels, which plays an important role in metabolic coupling and transport in lens fibers.

Original languageEnglish (US)
Pages (from-to)16914-16928
Number of pages15
JournalJournal of Biological Chemistry
Volume286
Issue number19
DOIs
StatePublished - May 13 2011

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Phosphorylation
Gap Junctions
Cyclic AMP-Dependent Protein Kinases
Coloring Agents
Connexins
Lenses
Phosphopeptides
Thin layer chromatography
Relocation
Thin Layer Chromatography
Alanine
Mass spectrometry
Chickens
Permeability
Mass Spectrometry
Chemical activation
Mutation
connexin 50
Fibers

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Phosphorylation of connexin 50 by protein kinase A enhances gap junction and hemichannel function. / Liu, Jialu; Ek Vitorin, Jose F; Weintraub, Susan T.; Gu, Sumin; Shi, Qian; Burt, Janis M; Jiang, Jean X.

In: Journal of Biological Chemistry, Vol. 286, No. 19, 13.05.2011, p. 16914-16928.

Research output: Contribution to journalArticle

Liu, Jialu ; Ek Vitorin, Jose F ; Weintraub, Susan T. ; Gu, Sumin ; Shi, Qian ; Burt, Janis M ; Jiang, Jean X. / Phosphorylation of connexin 50 by protein kinase A enhances gap junction and hemichannel function. In: Journal of Biological Chemistry. 2011 ; Vol. 286, No. 19. pp. 16914-16928.
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