Abstract
Purpose:
To identify the relationship between the PI3K signaling pathway and gap junction proteins to characterize its potential involvement in lens development and homeostasis.
Methods:
Electrophysiology experiments were performed using HeLa cells and Xenopus laevis oocytes using inhibition, or constitutive activation, of the PI3K signaling pathway to measure differences in gap junctional conductance between coupled cells. HeLa cells transfected with DNA encoding Connexin46 (Cx46) or Connexin50 (Cx50) were recorded by dual whole cell patch clamp in the presence of inhibitors of p110α (PIK75,50 nM) or its effector, Akt (Akt inhibitorVIII,10 µM). Gap junctional conductance was also measured using a Xenopus laevis oocyte expression system by dual whole cell voltage clamp of paired cells injected with cRNA. Recordings were taken from cells expressing connexins in the presence or absence of a constitutively active form of p110α (p110αH1047R). Lens epithelial cells were isolated from wild type and lens specific conditional p110α knockout mice. Cells were treated with quinine which specifically blocks Cx50 gap junctions and coupling was measured by dual whole cell patch clamp.
Results:
Treatment of HeLa cells with PIK75 reduced phospho-Akt levels, but not the total amount of Akt. There was no significant difference in gap junctional conductance in HeLa cells expressing Cx46 in the presence or absence of either PI3K inhibitor. There was a significant decrease in coupling in HeLa cells expressing Cx50 in the presence of the p110α or Akt inhibitors. In addition, voltage clamp experiments showed an increase in junctional conductance in oocyte pairs expressing both Cx50 and p110αH1047R cRNA, compared to those injected with Cx50 alone. Oocytes expressing p110αH1047R also showed a large increase in phospho-Akt levels. Lastly, when comparing wild type primary epithelial lens cells from those with a p110α deletion, p110α knockout cells showed a reduction in Cx50 coupling.
Conclusions:
Cx50 and Cx46 are differentially regulated by inhibition of p110α or Akt in transfected HeLa cells. Connexin 50 expressing Xenopus laevis oocytes have increased conductance when co-expressed with constitutively active p110α. Deletion of p110α in lens epithelial cells decreases Cx50 coupling. These data suggest that interaction between the PI3K signaling pathway and Cx50 may play a role in lens development or homeostasis.
Keywords: 532 gap junctions/coupling •
447 cell-cell communication •
446 cell adhesions/cell junctions