Mutations in Retinal Connexin35 in the Intracellular Loop or Carboxyl Terminus Block or Decrease Direct Phosphorylation by Protein Kinase A

Abstract

Abstract: : Purpose: Gap junctional coupling in some retinal neuronal networks can be regulated by dopamine-driven pathways involving cAMP and protein kinase A. Our previous work showed that PKA directly phosphorylates cytoplasmic domains of the neuronal gap junction protein, connexin35, in vitro. Our goal is to characterize the molecular basis for regulation of neuronal gap junctions by protein kinase A through in vitro and in vivo analyses. Methods: 6X-His tagged fusion proteins containing either the intracellular loop or carboxyl terminus of Cx35 were subjected to site-directed mutagenesis at multiple sites, eliminating either serine or threonine residues. The domains were expressed in E.coli, affinity purified, and phosphorylated in vitro with PKA and [γ-₃₃P] ATP. Phosphorylated proteins were resolved by SDS-PAGE and visualized with a phosphorimager. In vivo effects of the PKA pathway were studied in cultured cells transfected with Cx35. Following incubation with the PKA activator Sp-8-cpt-cAMPS or the competitive inhibitor Rp-8-cpt-cAMPS, tracer coupling was analyzed by Neurobiotin injection. Results: PKA directly phosphorylates Cx35 within the intracellular loop and the carboxyl terminal domains in vitro. For the intracellular loop, a mutation eliminating a single serine disrupted greater than 90% of the phosphorylation, compared to controls. For the carboxyl terminus, a single serine mutation greatly decreased phosphorylation compared to controls, and blocked it in the presence of a second mutated site. Cultured cells expressing Cx35 incubated with the PKA competitive inhibitor increased tracer coupling compared to levels for controls. Incubation with the PKA activator reduced coupling to levels below that for controls. Conclusions: Phosphorylation of Cx35 by PKA is complex: two major phosphorylation sites exist, one each in the intracellular loop and the carboxyl terminus. In addition, at least two minor sites are present. In vivo, activation or inhibition of PKA pathways modulates coupling in cells expressing Cx35. These results demonstrate that the decrease in coupling observed in Cx35 transfected cells could involve direct phosphorylation by PKA.

Keywords: 416 gap junctions/coupling • 515 phosphorylation • 525 protein modifications-post translational