Abstract
Recombinant type 3 ryanodine receptor (RyR3) has been purified in
quantities sufficient for structural characterization by cryoelectron
microscopy and three-dimensional (3D) reconstruction. Two cDNAs
were prepared and expressed in HEK293 cells, one encoding the wild-type
RyR3 and the other encoding RyR3 containing glutathione S-transferase
(GST) fused to its amino terminus (GST-RyR3). RyR3 was purified
from detergent-solubilized transfected cells by affinity chromatography
using 12.6-kDa FK506-binding protein in the form of a GST fusion
as the affinity ligand. Purification of GST-RyR3 was achieved by
affinity chromatography by using glutathione-Sepharose. Purified
recombinant RyR3 and GST-RyR3 proteins exhibited high-affinity
(3)Hryanodine binding that was sensitive to activation by Ca$^2+$
and caffeine and to inhibition by Mg$^2+$. 3D reconstructions
of both recombinant RyR3 and GST-RyR3 appeared very similar to
that of the native RyR3 purified from bovine diaphragm. Comparison
of the 3D reconstructions of RyR3 and GST-RyR3 revealed that the
GST domains and, hence, the amino termini of the RyR3 subunits
are located in the "clamp" structures that form the corners of the
square-shaped cytoplasmic region of homotetrameric RyR3. This study
describes the 3D reconstruction of a recombinant ryanodine receptor
and it demonstrates the potential of this technology for characterizing
functional and structural perturbations introduced by site-directed
mutagenesis.
- 11353864
- acid,
- adenosine
- alanine,
- animals,
- caffeine,
- calcium
- calcium,
- cell
- channel
- channel,
- co,
- conformation,
- cryoelectron
- dose-response
- drug,
- electrophysiology,
- fusion
- gating,
- gl,
- glutamic
- glutathione
- gov't,
- heart,
- humans,
- ion
- line,
- magnesium,
- mice,
- microscopy,
- muscle
- mutation,
- non-u.s.
- ntraction,
- p.h.s.,
- point
- protein
- proteins,
- receptor
- recombinant
- relationship,
- release
- research
- reticulum,
- ryanodine
- ryanodine,
- sarcoplasmic
- support,
- transfection,
- transferase,
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