Purpose: To determine the role of PKCγ in the regulation of gap junction coupling in the normal lens, we have compared the properties of coupling in lenses from wild type (WT) and PKC-γ knockout (KO) mice. Methods: Western blotting, confocal immunofluorescence microscopy, immunoprecipitation, RT-PCR and quantitative real time PCR were used to study gap junction protein and message expression; gap junction coupling conductance and pH gating were measured in intact lenses using impedance studies. Results: There were no gross differences in size, clarity, or expression of full-length Cx46 or Cx50 in lenses from WT and PKCγ KO mice. However, total Cx43 protein expression was ~150% higher in the KO lenses. In WT lenses, Cx43 was found only in epithelial cells whereas in KO lenses, its expression continued into the fiber cells. Gap junction coupling conductance in the differentiating fibers (DF) of PKCγ KO lenses was 34% larger than that of WT. In the mature fiber (MF), the effect was much larger with the KO lenses having an 82% increase in coupling over WT. pH gating of the DF fibers was not altered by the absence of PKCγ. Conclusion: PKCγ has a major role in the regulation of gap junction expression and coupling in the normal lens.
Bibliographical noteFunding Information:
This work is supported by NIH-EY-13421 to DJT, NIH-EY-06391 to RTM, NIH-P20-PRO17686 to COBRE Core B Kansas State University (KSU). Additional funding from the Johnson Center for Basic Cancer Research at KSU for the Summer Stipend to SD and Undergraduate Student Awards to LB and KM. We acknowledge the help in the lens dissections by Dr Vladimir Akoyev, the technical assistance of Dr Philine Wangemann and Joel D. Sanneman in acquiring the confocal images. We are grateful to Dr Xiaohua Gong for the Cx46 cytoplasmic loop antibody and drawing of the lens image by Sushanth Gudlur.
- Connexin 43
- Connexin 46
- PKCγKO mice
- Protein Kinase C gamma