PKA (protein kinase A)-dependent phosphorylation of the cardiac Ca2+-release channel/RyR2 (type 2 ryanodine receptor)is believed to directly dissociate FKBP12.6 (12.6 kDa FK506-binding protein) from the channel, causing abnormal channel activation and Ca2+ release. To gain insight into the structural basis of the regulation of RyR2 by PKA, we determined the three-dimensional location of the PKA site Ser2030. GFP (green fluorescent protein) was inserted into RyR2-wt (wild-type RyR2)and RyR2 mutant, A4860G, after Thr2023. The resultant GFP-RyR2 fusion proteins, RyR2T2023-GFP and RyR2(A4860G)T2023-GFP, were expressed in HEK-293 (human embryonic kidney) cells and functionally characterized. Ca2+-release assays revealed that both GFP-RyR2 fusion proteins formed caffeine- and ryanodine-sensitive Ca2+-release channels. Further analyses using[3H]ryanodine binding demonstrated that the insertion of GFPinto RyR2-wt after Thr2023 reduced the sensitivity of the channelto activation by Ca2+ or caffeine. RyR2(A4860G)T2023-GFP was found to be structurally more stable than RyR2T2023-GFP and was subsequently used as a basis for three-dimensional reconstruction. Cryo-electronmicroscopy and single particle image processing of the purified RyR2(A4860G)T2023-GFP protein revealed the location of the inserted GFP, and hence the Ser2030 PKA site in domain 4,a region that may be involved in signal transduction between the transmembrane and cytoplasmic domains. Like the Ser2808 PKA site reported previously, the Ser2030 site is not located close to the FKBP12.6-binding site mapped previously, indicating that neither of these PKA sites is directly involved in FKBP12.6 binding. On the basis of the three-dimensional localizations of a number of residues or regions, a model for the subunit organization in the structure of RyR2 is proposed.