The triad junction of skeletal muscle contains the components which transduce the signal of depolarization of the transverse tubule to evoke Ca2+ release from the sarcoplasmic reticulum. Two proteins which are recognized to participate in this pathway are the dihydropyridine receptor in the transverse tubule which senses the membrane potential and the ryanodine receptor in the terminal cisternae which is a Ca2+ release channel. The mechanism of communication between these two proteins is unknown. A third intrinsic protein of the triad junction, triadin, is in immediate juxtaposition to the ryanodine receptor in the sarcoplasmic reticulum and may form ternary complexes with the ryanodine receptor and the dihydropyridine receptor, providing the scaffolding for signal transduction. The overall goal of the research is to determine the architectural arrangement of the proteins of the triad junction and to determine the mechanism of physiological communication which accounts for muscle excitation. The organization of the proteins of the triad junction has proved difficult to study experimentally because the proteins are intrinsic proteins which are only dissolved from the membrane by detergent and hypertonic salt. In particular, triadin precipitates in isotonic media. To obviate these problems, the hydrophilic domains (both the cytoplasmic and the sarcoplasmic reticulum lumenal loops) of the ryanodine receptor, the dihydropyridine receptor and triadin will be expressed as fusion peptides with an engineered phosphate label. These fusion peptides provide an improved method for identifying and delimiting the interaction sites between the three known intrinsic proteins of the triad junction in physiological media in the absence of detergent. The probes will also provide a means of determining the intramolecular interactions of a loop of a given protein with other hydrophilic loops of itself and provide a tool to identify other proteins involved in the triad structure. In concert with these studies, the membrane topology of the ryanodine receptor and triadin will be determined using peptide directed antibodies to probe intact and partially permeabilized and/or proteolyzed triad vesicles. The fusion peptides will subsequently be used to probe for the physiological effects of the identified protein-protein interactions. The ability of the fusion peptides to alter ligand binding to the ryanodine and dihydropyridine receptors and to affect Ca2+ flux in intact vesicles or in isolated proteins will be tested. The converse experiment, the influence of channel state (activated or inactivated as induced by ligand binding) on the binding of the expressed peptide to intact vesicles or isolated proteins, will be employed to test for alterations in the pathway associated with signal transmission.
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