Z. Liu et al., Three-dimensional reconstruction of the recombinant type 3 ryanodine receptor and localization of its amino terminus, P NAS US, 98(11), 2001, pp. 6104-6109
Citations number
37
Categorie Soggetti
Multidisciplinary
Journal title
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Recombinant type 3 ryanodine receptor (RyR3) has been purified in quantitie
s sufficient for structural characterization by cryoelectron microscopy and
three-dimensional (3D) reconstruction. Two cDNAs were prepared and express
ed 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 for
m of a GST fusion as the affinity ligand, Purification of GST-RyR3 was achi
eved by affinity chromatography by using glutathione-Sepharose. Purified re
combinant RyR3 and GST-RyR3 proteins exhibited high-affinity [H-3]ryanodine
binding that was sensitive to activation by Ca2+ and caffeine and to inhib
ition by Mg2+. 3D reconstructions of both recombinant RyR3 and GST-RyR3 app
eared very similar to that of the native RyR3 purified from bovine diaphrag
m, 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 loca
ted in the "clamp" structures that form the corners of the square-shaped cy
toplasmic region of homotetrameric RyR3, This study describes the 3D recons
truction of a recombinant ryanodine receptor and it demonstrates the potent
ial of this technology for characterizing functional and structural perturb
ations introduced by site-directed mutagenesis.