ACTIVATION AND LABELING OF THE PURIFIED SKELETAL-MUSCLE RYANODINE RECEPTOR BY AN OXIDIZED ATP ANALOG

We have tested the periodate-oxidized ATP analogue 2',3'-dialdehyde ad enosine triphosphate (oATP) as a ligand for the skeletal muscle ryanod ine receptor/Ca2+-release channel. Ca2+ efflux from passively loaded h eavy sarcoplasmic reticulum vesicles of skeletal muscle is biphasic. o ATP stimulates the initial phase of Ca2+ release in a concentration-de pendent manner (EC(50) 160 mu M), and the efflux proceeds with a half- time in the range 100-200 ms. This oATP-modulated initial rapid Ca2+ r elease was specifically inhibited by millimolar concentrations of Mg2 and micromolar concentrations of Ruthenium Red, indicating that the e ffect of oATP was mediated via the ryanodine receptor. The purified Ca 2+-release channel was incorporated into planar lipid bilayers, and si ngle-channel recordings were carried out to verify a direct interactio n of oATP with the ryanodine receptor. Addition of oATP to the cytopla smic side activated the channel with an EC(50) of 76 mu M, which is ro ughly 30-fold higher than the apparent affinity of ATP. The oATP-induc ed increase in the open probability of the ryanodine receptor displays a steep concentration-response curve with a Hill coefficient of simil ar to 2, which suggests a co-operativity of the ATP binding sites in t he tetrameric protein. oATP binds to the ryanodine receptor in a quasi -irreversible manner via Schiff base formation between the aldehyde gr oups of oATP and amino groups in the nucleotide binding pocket. This a llows for the covalent specific incorporation of [alpha-P-32]oATP by b orhydride reduction. A typical adenine nucleotide binding site cannot be identified in the primary sequence of the ryanodine receptor. Our r esults demonstrate that oATP can be used to probe the structure and fu nction of the nucleotide binding pocket of the ryanodine receptor and presumably of other ATP-regulated ion channels.