Imaging of calcium sparks has provided a means for investigating calcium release from sarcoplasmic reticulum (SR) stores in intact muscle cells. Extraction of maximal information about the regulation and function of ryanodine receptor (RyR) SR calcium release channels requires that the calcium release flux underlying a spark be accurately quantified. Such quantification has proven difficult due to the complex nature of the cellular environment where sparks are imaged, and to the difficulty of manipulating single independent factors within an intact cell. Our goal is to implement a new, bottom-up approach to this problem. An optical system, optimized for imaging sparks in intact cells, will be used to obtain simple calcium dye signals to known calcium fluxes through single RyR channels under the controlled and relatively simple conditions of a planar lipid bilayer. The results of direct experimental evaluations of influences by cellular factors, such as calcium diffusion and binding by buffers, will be combined with the basic calcium bilayer signal to build up to the more complex waveform of a spark. To implement this approach we have developed an optical bilayer system capable of simultaneous measurement of calcium dye fluorescence and single RyR channel currents. Methods for immobilizing RyR channels in bilayers for continuous recording of optical signals related to single channel currents have been devised. In addition, a voltage-step protocol where RyR channel currents depend only on the imposed trans-bilayer voltage (which is known) and the ion concentrations in the cis and trans solutions (which are known) has been developed, implemented and tested. New insights will also be generated by the proposed studies. Not only will the details of the calcium flux underlying a spark be obtained, but also how individual cellular factors influence a spark will be dissected with direct measurements. In addition, the relationships between properties of the spark waveform and specific aspects of RyR channel function will be defined both qualitatively and quantitatively. Our approach is an experimental complement to current methods for modeling-simulation and will provide independent verification of model parameters and conclusions. We will measure empirically many of the factors that must be estimated using current methods.
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