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1 Molecular Biophysics and Physiology, Rush University, Chicago, Illinois, USA
2 Molecular Biophysics and Physiology, Rush University, Chicago, Illinois, USA; Biologia Experimental, Fundacion Instituto de Estudios Avanzados (IDEA), Caracas, Venezuela
3 Physiology, University of Illinois, Chicago, Illinois, USA
* To whom correspondence should be addressed. E-mail: erios{at}rush.edu.
To activate skeletal muscle contraction, action potentials must be sensed by dihydropyridine receptors (DHPRs) in the transverse (T) tubule, which signal the Ca2+ release channels (RyRs) in the sarcoplasmic reticulum (SR) to open. We demonstrate here an inhibitory effect of the T tubule on the production of sparks of Ca2+ release. Murine primary cultures were confocally imaged for Ca2+ detection and T tubule visualization. After 72 hrs of differentiation, T tubules extended from the periphery for less than 1/3 of the myotube radius. Spontaneous Ca2+ sparks were found away from the region of cells where tubules were found. Immunostaining showed RyR 1 and 3 isoforms in all areas, implying inhibition of both isoforms by a T tubule component. To test for a role of DHPRs in this inhibition, we imaged myotubes from dysgenic (mdg) mice, lacking DHPRs. These exhibited T tubule development similar to that of normal myotubes, but produced few sparks, even in regions where tubules were absent. To increase spark frequency a high Ca2+ saline with 1 mM caffeine was used. Wild-type cells in this saline plus 50 µM nifedipine retained the topographic suppression pattern of sparks, but dysgenic cells in high Ca2+ saline did not. SEER of indo-1 in the cytosol or mag-indo-1 in the SR was used to image [Ca2+] in these compartments. Under the conditions of interest, wild-type and mdg cells had similar levels of free [Ca2+] in cytosol and SR. These data suggest that DHPRs play a critical role in reducing the rate of spontaneous opening of Ca2+ release channels and/or their susceptibility to Ca2+-induced activation, thereby suppressing the production of Ca2+ sparks.
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