Abstract
Purpose: :
To determine the role of PKC-γ in regulation of gap junction expression and coupling in the normal lens, the properties of gap junction structure and coupling in lenses from wild type (WT) and PKC-γ knockout (KO) mice were compared.
Methods: :
Gap junction protein expression was determined by Western blotting, structure was observed using thin section and freeze fracture electron microscopy, and conical tomography, and coupling conductance was measured by impedance studies of intact lenses.
Results: :
There were no significant differences in size or expression of Cx46 or Cx50 in lenses from WT and PKC-γ KO mice. However, Cx43 was only found in epithelial cells of WT lenses, whereas expression continues into the fiber cells of PKCγ KO lenses. Outer differentiating fibers (DF) have the highest expression levels, but some protein could be detected in the inner mature fibers (MF). In PKCγ KO lenses, both DF and some fraction of MF contained vesicular like structures, formed from two cellular membranes connected by a gap junction. These structures could result from invaginations of cell to cell junctions that were sectioned and thus appeared vesicular, or from internalized junctions on their way to degradation. These structures were never seen in WT lenses. They represent a large increase in the area of gap junctions over that seen in WT lenses. DF gap junction coupling conductance GDF (S/cm2) was 0.31 for control vs 0.37 for KO; MF coupling conductance GMF (S/cm2) was 0.1 for control vs 0.25 for KO. Gap junction coupling conductance in the DF of PKC-γ KO lenses was therefore19% larger than that of WT, whereas in MF, the effect was much larger with the KO lenses having a 253% increase in coupling over WT.
Conclusions: :
PKC-γ regulates gap junction expression and coupling in the normal lens. The largest effects of PKC-γ KO on Cx43 expression and cellular structure occurred in the DF whereas the largest increase in coupling conductance occurred in the MF, where Cx46 is thought to be the only functional channel. The reason for the large increase in MF coupling is not known, as there appeared to be no significant increase in Cx46 protein; perhaps Cx46 is normally phosphorylated by PKC-γ, which inhibits the number of open channels.
Keywords: gap junctions/coupling • electrophysiology: non-clinical