Purchase this article with an account.
J. O'Brien, X. Ouyang, L.S. Patel, V.M. Winbow, G.S. Burr, C.K. Mitchell; PKA regulates connexin 35 gap junctions through a complex pathway . Invest. Ophthalmol. Vis. Sci. 2004;45(13):658.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: Connexin 35 (Cx35) is a major component of electrical synapses in the central nervous system. In the retina, many gap junctions containing Cx35 are regulated by dopamine receptor pathways involving cAMP–dependent protein kinase (PKA). We examined the mechanism by which PKA regulates Cx35. Methods: We identified phosphorylation sites through mutagenesis of predicted sites and in vitro phosphorylation of bacterially expressed Cx35–GST fusion proteins. We used quantitative analysis of Neurobiotin tracer coupling to study PKA regulation of mutant and wild–type Cx35 expressed in HeLa cells. Membrane–permeant cAMP analogs were used to inhibit or activate PKA. Results: Application of 20 µM Sp–8–cpt–cAMPS, a PKA activator, to Cx35–transfected HeLa cells caused a rapid decline in coupling rate that was maximal at 15 minutes. At 30 minutes, a large rebound in coupling was observed which could be blocked by the phosphatase inhibitor okadaic acid (100 nM). A PKA inhibitor, Rp–8–cpt–cAMPS (20 µM), increased coupling rate. In vitro phosphorylation showed that both intracellular loop and carboxyl terminal domains of Cx35 were phosphorylated. The mutation Ser110–Ala eliminated >95% of phosphorylation in the I–loop, while Ser128–Ala had little effect. In the C–terminal, Ser276–Ala eliminated >90% of phosphorylation, and truncation of the last 6 amino acids at Ser298 eliminated remaining phosphorylation. Each of the mutations Ser110–Ala, Ser276–Ala, and Ser298–ter significantly increased the coupling rate in HeLa cells in the absence of drugs compared to wild–type Cx35. Single mutations at Ser110 or Ser276 reduced the effects of the PKA activator and inhibitor. Truncation at Ser298 reversed the effects of cAMP analogs such that the PKA activator significantly increased and the PKA inhibitor significantly decreased coupling. Conclusions: PKA regulates Cx35 coupling in a complex and reversible manner. Phosphorylation of either Ser110 or Ser276 reduces coupling but both are needed to obtain significant regulation. However, phosphorylation of these residues enhances coupling in the absence of phosphorylation of or some other interaction with the C–terminal tip. Signaling through the latter site most likely involves a different effector since PKA phosphorylation here is minimal.
This PDF is available to Subscribers Only