In skeletal muscle sarcoplasmic reticulum (SR) a 106 kDa calcium release channel protein has been identified and purified through its "critical" free sulfhydryl by covalently linking it to biotin via a disulfide bond and using biotin-avidin chromatography. Upon incorporation in planar bilayers, this protein exhibited the properties of a calcium channel similar to those reported for the 400 kDa "feet" proteins. Immunological analysis using polyclonal antibodies against the 106 kDa and monoclonals against calcium- magnesium ATPase showed that the 106 kDa is distinct from SR calcium pumps and is neither a subunit nor a proteolytic fragment of the 400 kDa. The 106 kDa channels have been successfully purified by a) linear sucrose gradients, b) biotin avidin-, c) ryanodine, and d) immuno-affinity chromatography. The goals of the project are 1) To incorporate the purified 106 kDa calcium release channel protein into planar bilayers to characterize in detail the single channel ionic conductances, sub-states, selectivities and its modulation by agents that alter SR calcium release. The results will be compared with those reported for the 400 kDa "feet" protein. 2) To raise monoclonal antibodies against the 106 kDa channel and test them for possible modulation of calcium release in SR vesicles. 3) To localize this channel in junctional SR and rabbit psoas fibers by immuno-cytochemistry. 4) To re-examine the purification of the 400 kDa protein by linear sucrose gradients since the 106 kDa protein often co- migrates with the 400 kDa, and to use immuno-affinity columns to separate the 106 from the 400 kDa proteins. 5) Ryanodine- affinity columns have been made by 2 methods. Both columns bind selectively to the 106 kDa channel, implying that this channel has important physiological significance. The affinity and stoichiometry of tritiated ryanodine binding to 106 kDa will be measured and compared with binding data with junctional SR and the purified 400 kDa protein. 6) To determine the relationship and possible interaction of the 106 kDa channel with other SR proteins (i.e., the 400 kDa "feet" proteins) by covalently cross- linking the 106 kDa with neighboring junctional SR proteins using bi-functional cross-lining reagents of various spacer arm lengths. Recent progress in the understanding of excitation-contraction coupling has led to the general acceptance of the idea that the channel through which calcium is released from the sarcoplasmic reticulum is a 400 kDa protein which corresponds to structural "feet" seen in the electron microscope. Dr. Salama has accumulated substantial evidence, however, that a second, much smaller (106 kDa) protein may be the calcium release channel. The proposed experiments will characterize the smaller protein and elucidate its relationship to the "feet" protein. The results of this research should clarify the identity of the protein molecules responsible for calcium release from the sarcoplasmic reticulum in excitation-contraction coupling in striated muscle. ***//
