Selective phosphorylation of the IP3R-I in vivo by cGMP-dependent protein kinase in smooth muscle

This study examined the expression of inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) types and PKG isoforms in isolated gastric smooth muscle cells and determined the ability of PKG and PKA to phosphorylate IP(3)Rs and inhibit IP3-dependent Ca2+ release, which mediates the initial phase of agonist-induced contraction. PKG-Ialpha and PKG-Ibeta were expressed in gastric smooth muscle cells, together with IP3-R-associated cG-kinase substrate, a protein that couples PKG-Ibeta to IP3R-I. IP3R-I and IP3R-III were also expressed, but only IP3R-I was phosphorylated by PKA and PKG in vitro and exclusively by PKG in vivo. Sequential phosphorylation by PKA and by nPKG-Ialpha in vitro showed that PKA phosphorylated the same site as PKG (presumably S-1755) and an additional PKA-specific site (S-1589). In intact muscle cells, agents that activated PKG or both PKG and PKA induced IP3R-I phosphorylation that was reversed by the PKG inhibitor (8R,9S,11s)-(-)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,1H,-2,7b,11a-trizadizo-benzo9-(a,g) cycloocta(c,d,e)-trinden-1-one. Agents that activated PKA induced IP3R-I phosphorylation in permeabilized but not intact muscle cells, implying that PKA does not gain access to IP3R-I in intact muscle cells. The pattern of IP3R-I phosphorylation in vivo and in vitro was more consistent with phosphorylation by PKG-Ialpha. Phosphorylation of IP3R-I in microsomes by PKG, PKA, or a combination of PKG and PKA inhibited IP3-induced Ca2+ release to the same extent, implying that inhibition was mediated by phosphorylation of the PKG-specific site. We conclude that IP3R-I is selectively phosphorylated by PKG-I in intact smooth muscle resulting in inhibition of IP3-dependent Ca2+ release.