Identification and purification of a transverse tubule coupling protein which binds to the ryanodine receptor of terminal cisternae at the triad junction in skeletal muscle

Abstract

In fast twitch skeletal muscle, the signal for excitation-contraction coupling is transferred from transverse tubule across the triad junction; calcium is thereby released from the terminal cisternae of sarcoplasmic reticulum triggering muscle contraction. Recently, the feet structures of terminal cisternae, which bridge the gap at the triad junction, have been identified as the ryanodine receptor and in turn with the calcium release channels of sarcoplasmic reticulum. The latter consists of an oligomer of a single high molecular weight polypeptide (Mr 360,000). This study attempts to identify the component in the transverse tubule which ligands with the foot structure to form the triad junction. The purified ryanodine receptor, derivatized with sulfosuccinimidyl-2-(p-azidosalicylimido)-1,3'-dithiopropionate (SASD), a thiol-cleavable, 125I-iodinatable, and photoactive probe, was shown to selectively cross-link to a protein with Mr of 71,000 in isolated transverse tubules. This coupling protein was purified from transverse tubule by solubilization with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) and then purified by sequential column chromatography. In the absence of sulfhydryl agents, the purified polypeptide has an Mr of 61,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A complementary approach using SASD was employed to confirm association of the coupling protein with the ryanodine receptor of terminal cisternae. We conclude that the transverse tubule coupling protein together with the ryanodine receptor (foot structure) is involved in the liganding between transverse tubule and terminal cisternae of sacroplasmic reticulum.