Localization of calmodulin binding sites on the ryanodine receptor from skeletal muscle by electron microscopy.
Calmodulin (CaM) is a regulator of the calcium release channel (ryanodine receptor) of the sarcoplasmic reticulum of skeletal and cardiac muscle. The locations where CaM binds on the surface of the skeletal muscle ryanodine receptor were determined by electron microscopy. Wheat germ CaM was labeled specifically at Cys-27 with a maleimide derivative of a 1.4-nm-diameter gold cluster, and the gold-cluster-labeled CaM was bound to the purified ryanodine receptor. The complexes were imaged in the frozen-hydrated state by cryoelectron microscopy with no stains or fixatives present. In the micrographs, gold clusters were frequently observed near the corners of the square-shaped images of the ryanodine receptors. In some images, all four corners of the receptor were occupied by gold clusters. Image averaging allowed the site of CaM binding to be determined in two dimensions with an estimated precision of 4 nm. No changes were apparent in the quaternary structure of the ryanodine receptor upon binding CaM to the resolution attained, about 3 nm. Side views of the ryanodine receptor, in which the receptor is oriented approximately perpendicular to the much more frequent fourfold symmetric views, were occasionally observed, and showed that the CaM binding site is most likely on the surface of the receptor that faces the cytoplasm. We conclude that the CaM binding site is at least 10 nm from the transmembrane channel of the receptor and, consequently, that long-range conformational changes are involved in the modulation of the calcium channel activity of the receptor by CaM.
Wagenknecht
T
T
Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany 12201-0509.
Berkowitz
J
J
Grassucci
R
R
Timerman
A P
AP
Fleischer
S
S
D000818:Animals; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D015220:Calcium Channels / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D007091:Image Processing, Computer-Assisted; D066298:In Vitro Techniques; D008239:Lysine / Q000031:analogs & derivatives; D008301:Maleimides; D008854:Microscopy, Electron; D008958:Models, Molecular; D009124:Muscle Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D018482:Muscle, Skeletal / Q000378:metabolism* / Q000648:ultrastructure; D050607:Organogold Compounds; D009942:Organometallic Compounds; D011817:Rabbits; D019837:Ryanodine Receptor Calcium Release Channel
D015220:Calcium Channels; D002147:Calmodulin; D008301:Maleimides; D009124:Muscle Proteins; D050607:Organogold Compounds; D009942:Organometallic Compounds; D019837:Ryanodine Receptor Calcium Release Channel; C077615:monomaleimido gold; C047272:3-(N-maleimidopropionyl)biocytin; D008239:Lysine