Inhibitory phosphorylation of IP3 receptor type I (IP3R-I) In vivo is selectively mediated by cyclic GMP-dependent protein kinase-1α (PKG-1α) in gastric smooth muscle

Abstract

We have recently shown that PKG-Ia and PKG-I[3 are expressed in smooth muscle cells together with IP3 receptor-associated PKG substrate (IRAG). IRAG binds selectively to the N-terminal domain of PKG-I[3 and its phosphorylation at Ser696 by PKG-I~ is postulated to be essential for inhibition of [P3-induced Ca 2+ release by PKG. Both IP3Rol and IP3R-III were expressed in gastric smooth muscle cells, but only IP3R-I was phosphorylated by PKG in vivo leading to inhibition of IP~-dependent Ca 2+ release. In vitro phosphorylation of IP3RI in microsomal membranes by purified PKG-Ia holoenzyme inhibited IP3-induced Ca 2. release. Aim: To identify the PKG-I isoform responsible for inhibitory phosphorylation of IP3R-1 in vivo. Methods: Dominant negative PKG-In (T515A, T517A) and PKG-I~ (K405A) were expressed separately in cultured rabbit gastric smooth muscle cells. The ability of PKG to pbosphorylate IP3R-I and inhibit IP3-induced Ca 2§ release was determined in cells expressing both PKG-I isoforms and in cells expressing dominant negative PKG-Ia or PKG-IB. Results: Stimulation of PKG with sodium nitroprnsside (SNP I~M) or the nonhydrolyzable PKG activator, 8-pCPT--cGMP (10 g.M), caused phospboryiation (327 +_ 35% to 436 -+ 39% above basal level) of IP3R-I in muscle cells expressing both wild type PKG-I isoforms or in cells expressing dominant negative PKG-I[~, but not in cells expressing dominant negative PKG-Ia. IP3 stimulated Ca 2 + release in permeabilized muscle cells expressing wild type and mutant PKG-I isoforms to the same extent (29 -+ 3% to 33 + 4% decrease in steady-state ~5Ca2+ cell content). Treatment of the cells with SNP or 8-pCPT-cGMP inhibited IP3-induced Ca 2+ release in cells expressing both wild type PKG-I isoforms and in ceils expressing dominant negative PKG-I[3, but not in cells expressing dominant negative ?KG~Ia. The inhibition of lP3-indnced Ca 2+ release in cells expressing wild type and PKGI~ mutant was completely reversed by the PKG inhibitor, KT5823 (1 ~M). The results provide direct evidence that selective phosphorylation of IP~R-I by the PKG-Ia isoform mediates inhibition of IP3-induced Ca 2+ release in gastric smooth muscle cells. PKG-IB did not mediate phosphorylation of IP3R-I despite co-expression of IRAG in these cells. Conclusion: PKG-I~x mediates pbosphorylation of IP3R-1 and inhibition of IP3-induced Ca 2+ release.