Localization of PKA phosphorylation site, Ser²⁰³⁰, in the three-dimensional structure of cardiac ryanodine receptor Available to Purchase

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, RyR2_T2023-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 GFP into RyR2-wt after Thr²⁰²³ reduced the sensitivity of the channel to activation by Ca²⁺ or caffeine. RyR2(A4860G)_T2023-GFP was found to be structurally more stable than RyR2_T2023-GFP and was subsequently used as a basis for three-dimensional reconstruction. Cryo-electron microscopy 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.