Abstract
PKA (protein kinase A)-dependent phosphorylation of the cardiac Ca$^2+$-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 Ca$^2+$ 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.
Ca$^2+$-release assays revealed that both GFP-RyR2 fusion proteins
formed caffeine- and ryanodine-sensitive Ca$^2+$-release channels.
Further analyses using3Hryanodine binding demonstrated that the
insertion of GFPinto RyR2-wt after Thr2023 reduced the sensitivity
of the channelto activation by Ca$^2+$ 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.
- amp-dependent
- caffeine,
- calcium
- calcium,
- cell
- channel,
- chemistry/drug
- computer-assisted;
- conformation;
- cryoelectron
- cyclic
- effects/genetics/metabolism;
- effects/metabolism;
- embryology;
- enzymology;
- heart,
- humans;
- image
- kidney,
- kinases,
- line;
- metabolism/pharmacology;
- metabolism;
- microscopy;
- models,
- molecular;
- myocardium,
- nucleotide;
- pharmacology;
- phosphorylation;
- physiology;
- polymorphism,
- processing,
- protein
- proteins,
- receptor
- recombinant
- release
- ryanodine
- ryanodine,
- serine;
- single
- transfection
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