Abstract
The cardiac isoform of the ryanodine receptor (RyR2) from dog binds
predominantly a 12.6-kDa isoform of the FK506-binding protein (FKBP12.6),
whereas RyR2 from other species binds both FKBP12.6 and the closely
related isoform FKBP12. The role played by FKBP12.6 in modulating
calcium release by RyR2 is unclear at present. We have used cryoelectron
microscopy and three-dimensional (3D) reconstruction techniques to
determine the binding position of FKBP12.6 on the surface of canine
RyR2. Buffer conditions that should favor the öpen" state of RyR2
were used. Quantitative comparison of 3D reconstructions of RyR2
in the presence and absence of FKBP12.6 reveals that FKBP12.6 binds
along the sides of the square-shaped cytoplasmic region of the receptor,
adjacent to domain 9, which forms part of the four clamp (corner-forming)
structures. The location of the FKBP12.6 binding site on öpen" RyR2
appears similar, but slightly displaced (by 1-2 nm) from that found
previously for FKBP12 binding to the skeletal muscle ryanodine receptor
that was in the buffer that favors the "closed" state. The conformation
of RyR2 containing bound FKBP12.6 differs considerably from that
depleted of FKBP12.6, particularly in the transmembrane region and
in the clamp structures. The x-ray structure of FKBP12.6 was docked
into the region of the 3D reconstruction that is attributable to
bound FKBP12.6, to show the relative orientations of amino acid residues
(Gln-31, Asn-32, Phe-59) that have been implicated as being critical
in interactions with RyR2. A thorough understanding of the structural
basis of RyR2-FKBP12.6 interaction should aid in understanding the
roles that have been proposed for FKBP12.6 in heart failure and in
certain forms of sudden cardiac death.
- animals;
- binding
- binding;
- biophysics,
- blotting,
- calcium
- calcium,
- cell
- channel,
- chemistry/metabolism;
- chemistry;
- computer-assisted;
- conformation;
- cryoelectron
- dogs;
- electron;
- electrophoresis,
- factors;
- gel;
- image
- imaging,
- isoforms;
- membrane,
- metabolism;
- methods;
- microscopy,
- microscopy;
- microsomes,
- models,
- molecular
- molecular;
- muscle,
- myocardium,
- patch-clamp
- polyacrylamide
- processing,
- protein
- proteins,
- receptor
- recombinant
- release
- reticulum,
- ryanodine
- ryanodine,
- sarcoplasmic
- secondary;
- sites;
- skeletal,
- structure,
- tacrolimus
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