The PI of this proposal is an Instructor in Anesthesia at Brigham and Women's Hospital/Harvard Medical School. The work outlined in this proposal is intended to fund mentored training in two areas of muscle physiology to develop new skills in two cutting edge technologies. This mentored training will help the PI in the development of new working collaboration, leading to an application of R01 level funding and help in the transition of the PI from his current position to an independent academic investigator at Harvard Medical School. The mentors in this proposal are Dr. G. Stahl, who is an independent scientist with extensive experience in BIACORE analysis;Dr. K. Beam, an independent scientist with extensive experience in electrophysiology who is a renounce investigator in the field of muscle physiology and Dr. J. Lopez, an independent investigators with vast experience in microelectrode recording in muscle cells. The scientific work will be carried out at Harvard Medical School and UCHSC, two world-class scientific institutions where biomedical research is performed at the highest level. The long-term goal of the proposed study is to localize the structural determinant(s), within the primary sequence of skeletal ryanodine receptor (RyR1) and DHPR (1s and (1a subunit, involved in the direct interaction that these two channels engage on during skeletal muscle contraction and assess the functional effect of these interactions on RyR/DHPR signaling in vivo. Hypothesis: (1s and (1a DHPR subunits are linked to RyR1 through a complex physical interaction that requires multiple interacting points in both receptors. The domain(s) of RyR1 involved in these interactions are specific of RyR1 since homologous region(s) from non-coupled receptors (i.e. RyR3) do not support such interaction.
Specific Aim 1. To study the ability of DHPR oc1s and (5ia subunits to interact in vitro with wt-RyR1 and RyR3. Purified RyRs will be immobilized in BIACORE sensor chips capable of supporting lipid bilayer and interactions with DHPRs will be monitored by injecting either full-length a1s or p1a subunit over the activated surface.
Specific Aim 2. To define specific domains of RyR1 involved in the interaction with DHPR. We will study the binding properties of (1s and (1a to a library of chimeric RyR1/RyR3 receptors containing several regions RyR1 into a RyR3 background.
Specific Aim 3. To define the minimal essential domain of DHPR (1s and (1a involved in the physical interaction with RyR1. We will assay the ability of chimeric (1s/(1c subunits to interact in vitro with purified RyRs.
Specific Aim 4. To study the functional role of the identified (1s/RyR1 and (1a/RyR1 interactions on EC coupling and retrograde signaling and resting Ca2+. An extensive library of chimeric RyR3/RyR1 and DHPR (1s/(1c constructs will be expressed in single and double knockout myotubes (nulls for RyRs, DHPRs and RyR/DHPR) and tested using calcium imaging, patch-clamp and calcium microelectrodes. Relevance: A full understanding of the elaborate signaling processes that take place during skeletal muscle contraction has important implications for the development of new therapies aimed at improving contractility on pathophysiologic states.
