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 (ryanod ine receptor) of the sarcoplasmic reticulum of skeletal and cardiac mu scle. The locations where CaM binds on the surface of the skeletal mus cle ryanodine receptor were determined by electron microscopy. Wheat g erm 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 w as bound to the purified ryanodine receptor. The complexes were imaged in the frozen-hydrated state by cryoelectron microscopy with no stain s or fixatives present. In the micrographs, gold clusters were frequen tly observed near the corners of the square-shaped images of the ryano dine receptors. In some images, all four corners of the receptor were occupied by gold clusters. Image averaging allowed the site of CaM bin ding to be determined in two dimensions with an estimated precision of 4 nm. No changes were apparent in the quaternary structure of the rya nodine receptor upon binding CaM to the resolution attained, about 3 n m. Side views of the ryanodine receptor, in which the receptor is orie nted approximately perpendicular to the much more frequent fourfold sy mmetric views, were occasionally observed, and showed that the CaM bin ding 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 fr om the transmembrane channel of the receptor and, consequently, that l ong-range conformational changes are involved in the modulation of the calcium channel activity of the receptor by CaM.