Protein kinase A mediates regulation of gap junctions containing connexin35 through a complex pathway.
Connexin 35 (Cx35) is a major component of electrical synapses in the central nervous system. Many gap junctions containing Cx35 are regulated by dopamine receptor pathways that involve protein kinase A (PKA). To study the mechanism of PKA regulation, we analyzed direct phosphorylation of Cx35 by PKA in vitro and studied the regulation of neurobiotin tracer coupling in HeLa cells expressing Cx35 or Cx35 mutants that lack phosphorylation sites. In Cx35-transfected cells, application of the PKA activator Sp-8-cpt-cAMPS caused a significant decline in coupling, while a PKA inhibitor, Rp-8-cpt-cAMPS, significantly increased tracer coupling. In vitro phosphorylation and mutagenic analysis showed that PKA phosphorylates Cx35 directly at two major sites, Ser110 in the intracellular loop and Ser276 in the carboxyl terminus. In addition, a minor phosphorylation site in the C-terminus was identified by truncation of the last 7 amino acids at Ser298. The mutations Ser110Ala or Ser276Ala significantly reduced regulation of coupling by the PKA activator while a combination of the two eliminated regulation. Truncation at Ser298 reversed the regulation such that the PKA activator significantly increased and the PKA inhibitor significantly decreased coupling. The activation was eliminated in the S110A, S276A, S298ter triple mutant. We conclude that PKA regulates Cx35 coupling in a complex manner that requires both major phosphorylation sites. Furthermore, the tip of the C-terminus acts as a "switch" that determines whether phosphorylation will inhibit or enhance coupling. Reliance on the combined states of three sites provides fine control over the degree of coupling through Cx35 gap junctions.
Ouyang
Xiaosen
X
Department of Ophthalmology and Visual Science, University of Texas, Houston Health Science Center, 6431 Fannin Street, MSB 7.024, Houston, TX 77030, USA.
Winbow
Virginia M
VM
Patel
Leena S
LS
Burr
Gary S
GS
Mitchell
Cheryl K
CK
O'Brien
John
J
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D017630:Connexins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017629:Gap Junctions / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D051379:Mice; D008954:Models, Biological; D016296:Mutagenesis / Q000502:physiology; D009154:Mutation; D010471:Perches; D010766:Phosphorylation / Q000187:drug effects; D012333:RNA, Messenger / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D016415:Sequence Alignment; D012694:Serine / Q000235:genetics / Q000378:metabolism; D012860:Skates, Fish; D014162:Transfection / Q000379:methods
D017630:Connexins; D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C074155:8-chloroadenosine-3',5'-cyclic monophosphorothioate; D012694:Serine; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D000409:Alanine