title: Phosphorylation of connexin 50 by protein kinase A enhances gap junction and hemichannel function.
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.
authors: Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229, USA.
authors: Jialu
authors: J
authors: Liu
authors: Jose F
authors: JF
authors: Ek Vitorin
authors: Susan T
authors: ST
authors: Weintraub
authors: Sumin
authors: S
authors: Gu
authors: Qian
authors: Q
authors: Shi
authors: Janis M
authors: JM
authors: Burt
authors: Jean X
authors: JX
authors: Jiang
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002642:Chick Embryo; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002855:Chromatography, Thin Layer / Q000379:methods; D017630:Connexins / Q000737:chemistry*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D005136:Eye Proteins / Q000737:chemistry*; D017629:Gap Junctions / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods; D010748:Phosphopeptides / Q000737:chemistry; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000737:chemistry; D012694:Serine / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D014357:Trypsin / Q000737:chemistry; D000099264:Connexin 50
chemical_list: D017630:Connexins; D017868:Cyclic AMP-Dependent Protein Kinases; D005136:Eye Proteins; D010748:Phosphopeptides; D011994:Recombinant Proteins; D012694:Serine; D014357:Trypsin; D000099264:Connexin 50