Abstract
Abstract: :
Purpose: Gap junctions in many retinal neurons are regulated by nitric oxide (NO), presumably through activation of guanylyl cyclase (sGC) and subsequent activation of cGMP–dependent protein kinase (PKG). This study is intended to elucidate the molecular mechanisms of this regulation in Cx35, the most widespread connexin in retinal neurons. Methods:Tracer coupling assays in HeLa cells expressing wild–type or mutant Cx35. In vitro phosphorylation and tandem mass spectroscopy analysis of phosphorylated peptides. Results: Application of the NO donor spermine NONOate (1 to 100 µM) caused significant uncoupling of HeLa cells expressing Cx35. Spermine alone had no effect. Activation of sGC in the absence of exogenous NO using 5 µM Bay 41–2272 also significantly reduced coupling, indicating that elevation of cGMP reduces coupling. However, inhibition of sGC with 5 µM ODQ significantly reduced coupling as well. This suggests that a pathway dependent on constitutive synthesis of cGMP is required to maintain coupling. In vitro phosphorylation of Cx35 with PKG and peptide analysis by tandem mass spectroscopy showed that PKG phosphorylated Cx35 at three sites: Ser110, Ser276, and Ser289. We have previously shown that PKA phosphorylates Ser110 and Ser276, and that phosphorylation at both sites together reduces coupling. To analyze the influence of phosphorylation on Ser289, we examined tracer coupling in HeLa cells expressing a S289A mutant. S289A HeLa cells were coupled similarly to wild type Cx35 cells, and neither Bay 41–2272 nor ODQ altered coupling. Thus phosphorylation of Ser289 is required for both activation and inactivation effects. Conclusions: Cx35 is subject to regulation by nitric oxide and cGMP–dependent pathways in a complex manner. Phosphorylation of Ser289 can contribute to either opening or closing of channels, but the full regulatory behavior probably depends on the phosphorylation states of several residues.
Keywords: gap junctions/coupling • nitric oxide • phosphorylation