%0 Journal Article %1 Holl_2000_551 %A Hollingworth, S. %A Soeller, C. %A Baylor, S. M. %A Cannell, M. B. %D 2000 %J J. Physiol. %K Acid Acid, Action Adaptation, Adenosine Adhesion, Algorithms, Amino Aniline Animals, Antibodies, Antigens, Bilayers, Biological, Biosensing Butyric C57BL, Cadherins, Calcium Calcium, Cardiac, Cardiovascular, Carrier Cell Cells, Channel Channels, Communication, Comparative Compartmentation, Compounds, Computer Computer-Assisted, Conductivity, Confocal, Connexins, Crystalline, Crystallins, Culture Cultured, Dermatophagoides, Desmosomes, Diagnostic Differentiation, Diffusion, Dogs, Dyes, Electric Electrochemistry, Electrophysiology, Enhancement, Epithelial Epitopes, Feasibility Feces, Feedback, Fluorescein, Fluorescein-5-isothiocyanate, Fluorescence, Fluorescent Gap Gating, Glycoproteins, Heart, Humans, Hybridization, I, Image Imaging, In Inbred Ion Ions, Junctions, Kinetics, L-Type, Lasers, Lens, Line, Lipid Membrane Membrane, Messenger, Mice, Microscopy, Mites, Models, Permeability, Potentials, Processing, Proteins, Sequence, Signaling, Simulation, Situ Stimulation, Studies, Study, Techniques, Triphosphate, Vitro, %P 551-60 %T Sarcomeric Ca$^2+$ gradients during activation of frog skeletal muscle fibres imaged with confocal and two-photon microscopy. %U http://jp.physoc.org/cgi/content/full/526/3/551 %V 526 Pt 3 %X Intra-sarcomeric gradients of Ca$^2+$ during activation of action potential stimulated frog single fibres were investigated with the Ca$^2+$ indicator fluo-3 and confocal and two-photon microscopy. The object of these experiments was to look for evidence of extra-junctional Ca$^2+$ release and examine the microscopic diffusion of Ca$^2+$ within the sarcomere. By exploiting the spatial periodicity of sarcomeres within the fibre, we could achieve a high effective line-scanning rate ( approximately 8000 lines s-1), although the laser scanning microscope was limited to < 1000 lines s-1. At this high time resolution, the time course of fluorescence changes was very different at the z- and m-lines, with a significant delay ( approximately 1 ms; 22 C) between the rise of fluorescence at the z-line and the m-line. To calculate the expected fluorescence changes, we used a multi-compartment model of Ca$^2+$ movements in the half-sarcomere in which Ca$^2+$ release was restricted to triadic junctions (located at z-lines). Optical blurring by the microscope was simulated to generate fluorescence signals which could be compared directly to experimental data. The model which reproduced our experimental findings most accurately included Ca$^2+$ binding by ATP, as well as indicator binding to immobile sarcomeric proteins. After taking sarcomeric misregistration within the fibre into account, there was very good agreement between the model and experimental results. We conclude that there is no experimental evidence for Ca$^2+$ release at locations other than at z-lines. In addition, our calculations support the conclusion that rapidly diffusing Ca$^2+$ buffers (such as ATP) are important in shaping the Ca$^2+$ transient and that the details of intracellular indicator binding need to be considered to explain correctly the time course of fluorescence change in the fibre.

@article{Holl_2000_551, abstract = {Intra-sarcomeric gradients of [{C}a$^{2+}$] during activation of action potential stimulated frog single fibres were investigated with the {C}a$^{2+}$ indicator fluo-3 and confocal and two-photon microscopy. The object of these experiments was to look for evidence of extra-junctional {C}a$^{2+}$ release and examine the microscopic diffusion of {C}a$^{2+}$ within the sarcomere. By exploiting the spatial periodicity of sarcomeres within the fibre, we could achieve a high effective line-scanning rate ( approximately 8000 lines s-1), although the laser scanning microscope was limited to < 1000 lines s-1. At this high time resolution, the time course of fluorescence changes was very different at the z- and m-lines, with a significant delay ( approximately 1 ms; 22 C) between the rise of fluorescence at the z-line and the m-line. To calculate the expected fluorescence changes, we used a multi-compartment model of {C}a$^{2+}$ movements in the half-sarcomere in which {C}a$^{2+}$ release was restricted to triadic junctions (located at z-lines). Optical blurring by the microscope was simulated to generate fluorescence signals which could be compared directly to experimental data. The model which reproduced our experimental findings most accurately included {C}a$^{2+}$ binding by ATP, as well as indicator binding to immobile sarcomeric proteins. After taking sarcomeric misregistration within the fibre into account, there was very good agreement between the model and experimental results. We conclude that there is no experimental evidence for {C}a$^{2+}$ release at locations other than at z-lines. In addition, our calculations support the conclusion that rapidly diffusing {C}a$^{2+}$ buffers (such as ATP) are important in shaping the {C}a$^{2+}$ transient and that the details of intracellular indicator binding need to be considered to explain correctly the time course of fluorescence change in the fibre.}, added-at = {2009-06-03T11:20:58.000+0200}, author = {Hollingworth, S. and Soeller, C. and Baylor, S. M. and Cannell, M. B.}, biburl = {https://www.bibsonomy.org/bibtex/2ff770fb5b4e99bcd484a27a30eef9d72/hake}, description = {The whole bibliography file I use.}, file = {Holl_2000_551.pdf:Holl_2000_551.pdf:PDF}, interhash = {ca3cea1454b9d110397585a2b0be08a4}, intrahash = {ff770fb5b4e99bcd484a27a30eef9d72}, journal = {J. Physiol.}, keywords = {Acid Acid, Action Adaptation, Adenosine Adhesion, Algorithms, Amino Aniline Animals, Antibodies, Antigens, Bilayers, Biological, Biosensing Butyric C57BL, Cadherins, Calcium Calcium, Cardiac, Cardiovascular, Carrier Cell Cells, Channel Channels, Communication, Comparative Compartmentation, Compounds, Computer Computer-Assisted, Conductivity, Confocal, Connexins, Crystalline, Crystallins, Culture Cultured, Dermatophagoides, Desmosomes, Diagnostic Differentiation, Diffusion, Dogs, Dyes, Electric Electrochemistry, Electrophysiology, Enhancement, Epithelial Epitopes, Feasibility Feces, Feedback, Fluorescein, Fluorescein-5-isothiocyanate, Fluorescence, Fluorescent Gap Gating, Glycoproteins, Heart, Humans, Hybridization, I, Image Imaging, In Inbred Ion Ions, Junctions, Kinetics, L-Type, Lasers, Lens, Line, Lipid Membrane Membrane, Messenger, Mice, Microscopy, Mites, Models, Permeability, Potentials, Processing, Proteins, Sequence, Signaling, Simulation, Situ Stimulation, Studies, Study, Techniques, Triphosphate, Vitro,}, month = Aug, pages = {551-60}, pii = {PHY_0639}, timestamp = {2009-06-03T11:21:15.000+0200}, title = {Sarcomeric {C}a$^{2+}$ gradients during activation of frog skeletal muscle fibres imaged with confocal and two-photon microscopy.}, url = {http://jp.physoc.org/cgi/content/full/526/3/551}, volume = {526 Pt 3}, year = 2000 }