An integrated effort is proposed to measure specific signals in the excitation-contraction (E-C) coupling sequence in vertebrate striated muscle and to relate these signals in a quantitative fashion to the release and re-uptake of calcium ions from the sarcoplasmic reticulum (SR). The overall goal is to attain a detailed understanding of the mechanisms that function in living muscle fibers to control the rapid changes in intracellular calcium that in turn regulate the normal contractile response. The major technical approach involves the measurement of the cytoplasmic free concentration of calcium ions (CA), magnesium ions (Mg) and protons (H) in resting skeletal muscle fibers and changes in these levels in response to electrical stimulation. The experimental design involves the micro-injection of single muscle cells with """"""""indicator"""""""" dyes that report optical signals (e.g. absorbance or fluorescence changes) that are specific for specific ions -- for example, Antipyrylazo III and/or Fura-2 for Ca, Phenol Red for H, Furaptra for Mg. In some experiments two dyes specific for two different ions will be employed. The quantitative interpretation of the results depends on: (a) in vitro dye calibrations carried out in the absence and presence of muscle constituents and (b) a computational model which incorporates published biochemical data about the principal ion binding sites in myoplasm and anatomical data about the structure of a sarcomere. The experiments will aim to determine (i) what physiological """"""""message"""""""" links electrical activity in the transverse tubular membranes with SR Ca- releasing activity; (ii) how myoplasmic levels of Ca, Mg and H affect the rate at which Ca is released from and re-sequestered into the SR; (iii) the extent to which H, Mg and K may serve as electrical """"""""counter-ions"""""""" to Ca movements during the release process; (iv) what change in SR membrane potential may accompany SR Ca release; (v) how Ca binding to troponin an parvalbumin are related to myofilament activation and relaxation, respectively; (vi) whether Ca binding to a calmodulin-like receptor molecule in myoplasm mediates Ca-inhibition of SR Ca release; (vii) what spatial differences in [Ca] may be expected within the sarcomere during the activation process and whether these differences can be detected experimentally be useful for understanding how specific events in muscle performance are altered by drugs and disease.
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