Figure 7. Hypothetical model for Cx35 regulation.

A. Schematic summary of the nitric oxide signaling system in HeLa cells. NO acts through two distinct pathways, one depending on the activity of guanylyl cyclase and the other independent of this activity. The pathways converge to uncouple Cx35 gap junctions through a mechanism that requires PKA activity. cGMP can either activate or inhibit different isoforms of phosphodiesterase (PDEs), thereby regulating local cAMP concentration. The cGMP-inhibited PDE3 could account for uncoupling (but see discussion). Activation of PKG by cGMP could result in phosphorylation of small polypeptide inhibitors of protein phosphatases (PPase) such as G-substrate (GS) or DARPP-32, and subsequent inhibition of phosphatase activity. PKG could also activate PKA through an unknown mechanism. It is unclear if PKG phosphorylates Cx35 directly. In the cGMP-independent pathway, NO may activate PKA directly, may inhibit a protein phosphatase activity, or may sensitize Cx35 to the action of PKA.

B. Summary of the phosphorylation sites on Cx35 that regulate its coupling. Ser110 in the intracellular loop and Ser276 in the C-terminal domain are key targets of phosphorylation that uncouple the gap junctions. Both PKA and PKG can phosphorylate these sites. Other sites in the outer half of the C-terminus such as Ser289 have weaker effects, but may modulate the effects of phosphorylation on the two major sites.