(Support NIH/NCRR/P-41 RR 01219 and NIH AR40615 to T. Wagenknecht). In both skeletal and heart muscles, the key events of muscle contraction occur at subcellular structures known as triad junctions. The detailed three-dimensional architecture of the triad junction will contribute to better understanding of the molecular basis of muscle contraction. This understanding may ultimately lead to improved treatments for diseases that affect the muscular contraction process. In skeletal muscle, these diseases include malignant hyperthermia, central core disease, and hypokalemic periodic paralysis. Many of the drugs that are currently used to treat heart disease are believed to produce therapeutic effects by acting on components of the triad junction. Understanding the structure of the triad junction is critical to design highly effective drugs for heart diseases. The structure of the frog muscle triad junction is being studied to investigate the site of the calcium release channel in relation to the t-tubes and sarcoplasmic reticulum. This is complementary to the reconstructions of the calcium release channel being done in Dr. Wagenknecht's group using the single-particle approach. In the BMIRR grant proposal, the triad junction was designated as one of the three objects (the others were the mitochondrion and the primary cilium) which would be used for development and testing of the automated tomography system. In the previous reporting period, four double-tilt reconstructions of triad junctions from high-pressure frozen, freeze-substituted frog sartorius muscle were made from 0.18(m plastic sections, using the IVEM. A reconstruction was also made from a frozen-hydrated preparation of isolated triad junctions. The work was reported at several meetings. During this reporting period, the previously obtained tilt-series data and reconstructions were used to test different weighting schemes for 3-D reconstruction, and the usefulness of """"""""wavelet"""""""" denoising techniques (see TRD """"""""Use of wavelets for noise reduction of images for tomography""""""""). New reconstructions from the old tilt-series images were made by the weighted back-projection method, using a weighting function for arbitrary tilt geometry (M. Radermacher (1992) Weighted back-projection methods. In: Electron Tomography, Ed., J Frank, Plenum, New York). These were compared to reconstructions made with the R-weighted back-projection method. Reconstructions were made with and without wavelet-based denoising techniques. The results are now being analyzed.
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