Excitation-contraction (EC) coupling is the process that translates the action potential in a muscle cell to increase in intracellular [Ca2+] and contraction. Our goal is to understand the cell-wide calcium signal of EC coupling in terms of contributions from individual calcium channels (RyRs) in the membrane of the storage organelle. The native environment imposes structural constraints and interactions of great complexity among RyRs in the same membrane, as well as with proteins in the T membrane or inside the store. In 1995 we described in amphibians a dramatic local response first seen in the heart, the Ca2+ spark, and showed it to require opening of multiple channels. We and others saw this as resulting from interactions mediated by calcium itself, which may cause channel opening (CICR) or inactivation (GDI). More recently we found that mammals respond with """"""""embers"""""""", one-channel events. We hypothesize that different events reflect different RyR isoforms and geometry in these taxa, which impose differences in CICR and GDI. We will test the hypothesis probing the presence and properties of CICR and GDI by direct application of local Ca2+ or indirectly by changing store content, as we image in parallel [Ca2+] in the cytosol and inside the store. To further separate the effects of isoforms and geometry, this will be done in adult or developing cells engineered to alter isoform composition and geometry, including for the first time adult mice with a muscle gene transiently silenced by RNA interference and others re-expressing a protein that is suppressed in postnatal development. Crucial techniques include innovative regional release from """"""""caged Ca2+"""""""" and a new method to image [Ca2+] in organelles that we named SEER. For transfer of large DMA code into adult cells we implemented an electroporation method of DiFranco and colleagues (2005), and a number of viral procedures, including helper virus-free amplicon transduction. Calcium signals are universal, but it is in muscle that they are fastest and most massive. Muscle's devices continue to be watched as possible paradigms for neurons, nuclear gene-regulatory signals, and linkers of store-operated plasmalemmal channels to intracellular sensors. While we work on fundamentals of normal function in health, these mechanisms are basic to three types of functional loss, in diseases of EC coupling, short term muscle fatigue, and in the aging process. ? ? ? ?
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