Regulation of lens cell-to-cell communication by activation of PKCgamma and disassembly of Cx50 channels.
OBJECTIVE
Lens fiber gap junctions comprise approximately equal molar amounts of connexin46 (Cx46) and connexin50 (Cx50), both of which contribute significantly to coupling in the lens cortex and nucleus. The current study was conducted to test the hypothesis that regulation of lens coupling by activation of protein kinase Cgamma (PKCgamma) affects the number of channels composed of Cx46, Cx50, or both connexins.


METHODS
Whole rat lenses were treated with phorbol-12-myristate-13-acetate (TPA) to activate PKCgamma or the inactive analogue 4alpha-phorbol,12,13-didecaneote (PDD) as a control. The superficial cortical fibers were studied morphologically by quantitative freeze-fracture immunolabeling (FRIL); functionally by Lucifer yellow dye transfer assay; and chemically by measuring PKCgamma activity, connexin phosphorylation and coimmunoprecipitation.


RESULTS
Treatment with TPA activated PKCgamma and uncoupled the lens cortex by approximately 60%. PDD had no effect. Activation of PKCgamma decreased the density of Cx50 channels assembled in gap junctions, increased the density of Cx50 hemichannels in the plasma membrane and induced circular voids measuring 22 to 300 nm in diameter within the remaining plaques. Coimmunoprecipitation studies indicated that the soluble PKCgamma was translocated into membrane fractions that contained Cx46, Cx50, and the lipid raft marker caveolin (Cav)-1. In the membrane environment, PKCgamma phosphorylated Cx50 at serines and threonines and Cx46 only at threonines.


CONCLUSIONS
The studies provide experimental support for the hypothesis that gap junctions comprising mixtures of Cx46 and Cx50 channels provide malleable communicating pathways between the lens nucleus and the metabolically active fibers in the surface. The findings also suggest that Cx50 channel disassembly occurs in distinct lipid microdomains.
Zampighi
Guido A
GA
Department of Neurobiology and Jules Stein Eye Institute, David Geffen School of Medicine, University of California-Los Angeles, Los Angeles, CA 90095, USA. gzampighi@mednet.ucla.edu
Planells
Ana M
AM
Lin
Dingbo
D
Takemoto
Dolores
D
D000818:Animals; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism; D002450:Cell Communication / Q000187:drug effects / Q000502:physiology*; D017630:Connexins / Q000378:metabolism*; D004789:Enzyme Activation; D005136:Eye Proteins / Q000378:metabolism*; D005260:Female; D005614:Freeze Fracturing; D017629:Gap Junctions / Q000378:metabolism* / Q000648:ultrastructure; D007473:Ion Channels / Q000378:metabolism* / Q000648:ultrastructure; D007546:Isoquinolines / Q000378:metabolism; D007908:Lens, Crystalline / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D000099264:Connexin 50
D051242:Caveolin 1; D022461:Caveolins; D017630:Connexins; D005136:Eye Proteins; D007473:Ion Channels; D007546:Isoquinolines; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D000099264:Connexin 50; C495537:Cav1 protein, rat; C487370:Gja3 protein, rat; C017475:lucifer yellow; C087857:protein kinase C gamma