Calmodulin and protein kinase C regulate gap junctional coupling in lens epithelial cells

Area of Science:

• Ophthalmology
• Cell Biology
• Physiology

Background:

• Lens epithelial cells communicate via gap junctions.
• Gap junction permeability is regulated by intracellular signaling pathways.
• Cytosolic calcium ion (Ca2+) and protein kinase C (PKC) are implicated in regulating cell communication.

Purpose of the Study:

• To investigate the distinct mechanisms of lens epithelial cell gap junction regulation by calcium ionophore and ATP.
• To differentiate the roles of calmodulin (CaM) and PKC in mediating these signaling pathways.

Main Methods:

• Utilized inhibitors of calmodulin (CaM) and PKC to block specific signaling pathways.
• Measured cell-to-cell transfer of fluorescent dye (AlexaFluor594) to assess gap junction permeability.
• Manipulated cytosolic Ca2+ (Cai2+) levels using calcium ionophore and ATP agonists.
• Employed BAPTA to buffer intracellular Ca2+.

Main Results:

• Calcium ionophore-induced sustained increase in Cai2+ inhibited gap junctions via CaM, not PKC.
• ATP-induced delayed, transient decrease in cell coupling was mediated by PKC and independent of Cai2+ changes.
• No alteration in connexin43 phosphorylation was observed during ATP-induced coupling decrease.
• BAPTA-loaded cells still exhibited ATP-dependent coupling decrease, confirming Cai2+ independence.

Conclusions:

• CaM mediates lens gap junction inhibition under sustained, high cytosolic Ca2+ conditions.
• PKC mediates lens gap junction inhibition through Ca2+-independent pathways activated by ATP.
• Distinct signaling cascades involving CaM and PKC differentially regulate lens epithelial cell gap junction communication.