A probable role of dihydropyridine receptors in repression of Ca2+ sparks demonstrated in cultured mammalian muscle.
To activate skeletal muscle contraction, action potentials must be sensed by dihydropyridine receptors (DHPRs) in the T tubule, which signal the Ca(2+) release channels or ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) to open. We demonstrate here an inhibitory effect of the T tubule on the production of sparks of Ca(2+) release. Murine primary cultures were confocally imaged for Ca(2+) detection and T tubule visualization. After 72 h of differentiation, T tubules extended from the periphery for less than one-third of the myotube radius. Spontaneous Ca(2+) sparks were found away from the region of cells where tubules were found. Immunostaining showed RyR1 and RyR3 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 mice (mdg) that lack 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-Ca(2+) saline with 1 mM caffeine was used. Wild-type cells in this saline plus 50 microM nifedipine retained the topographic suppression pattern of sparks, but dysgenic cells in high-Ca(2+) saline did not. Shifted excitation and emission ratios of indo-1 in the cytosol or mag-indo-1 in the SR were used to image [Ca(2+)] in these compartments. Under the conditions of interest, wild-type and mdg cells had similar levels of free [Ca(2+)] in cytosol and SR. These data suggest that DHPRs play a critical role in reducing the rate of spontaneous opening of Ca(2+) release channels and/or their susceptibility to Ca(2+)-induced activation, thereby suppressing the production of Ca(2+) sparks.
Zhou
Jingsong
J
Section of Cellular Signaling, Department of Molecular Biophysics and Physiology, Rush University, Chicago 60612, USA. jzhou1@rush.edu
Yi
Jianxun
J
Royer
Leandro
L
Launikonis
Bradley S
BS
González
Adom
A
García
Jesús
J
Ríos
Eduardo
E
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D002478:Cells, Cultured; D005456:Fluorescent Dyes / Q000378:metabolism; D051379:Mice; D009119:Muscle Contraction / Q000502:physiology; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000378:metabolism; D018482:Muscle, Skeletal / Q000166:cytology / Q000254:growth & development / Q000502:physiology; D020033:Protein Isoforms / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism / Q000648:ultrastructure
D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D005456:Fluorescent Dyes; D020033:Protein Isoforms; D019837:Ryanodine Receptor Calcium Release Channel; D002118:Calcium