Differential localization and functional role of calsequestrin in growing and differentiated myoblasts.
Calsequestrin (CSQ) is the low affinity, high capacity Ca(2+)-binding protein concentrated within specialized areas of the muscle fiber sarcoplasmic reticulum (a part of the ER) where it is believed to buffer large amounts of Ca2+. Upon activation of intracellular channels this Ca2+ pool is released, giving rise to the [Ca2+]i increases that sustain contraction. In order to investigate the ER retention and the functional role of the protein, L6 rat myoblasts were infected with a viral vector with or without the cDNA of chicken CSQ, and stable clones were investigated before and after differentiation to myotubes. In the undifferentiated L6 cells, expression of considerable amounts of heterologous CSQ occurred with no major changes of other ER components. Ca2+ release from the ER, induced by the peptide hormone vasopressin, remained however unchanged, and the same occurred when other treatments were given in sequence to deplete the ER and other intracellular stores: with the Ca2+ pump blocker, thapsigargin; and with the Ca2+ ionophore, ionomycin, followed by the Na+/H+ ionophore, monensin. The lack of effect of CSQ expression on the vasopressin-induced [Ca2+]i responses was explained by immunocytochemistry showing the heterologous protein to be localized not in the ER but in large vacuoles of acidic content, positive also for the lysosomal enzyme, cathepsin D, corresponding to a lysosomal subpopulation. After differentiation, all L6 cells expressed small amounts of homologous CSQ. In the infected cells the heterologous protein progressively decreased, yet the [Ca2+]i responses to vasopressin were now larger with respect to both control and undifferentiated cells. This change correlated with the drop of the vacuoles and with the accumulation of CSQ within the ER lumen, where a clustered distribution was observed as recently shown in developing muscle fibers. These results provide direct evidence for the contribution of CSQ, when appropriately retained, to the Ca2+ capacity of the rapidly exchanging, ER-located Ca2+ stores; and for the existence of specific mechanism(s) (that in L6 cells develop in the course of differentiation) for the ER retention of the protein. In the growing L6 myoblasts the Ca(2+)-binding protein appears in contrast to travel along the exocytic pathway, down to post-Golgi, lysosome-related vacuoles which, based on the lack of [Ca2+]i response to ionomycin-monensin, appear to be incompetent for Ca2+ accumulation.
Department of Pharmacology, CNR Molecular and Cellular Pharmacology, Scientific Institute San Raffaele, University of Milano, Italy.
M
M
Raichman
M C
MC
Panzeri
E
E
Clementi
P
P
Papazafiri
M
M
Eckley
D O
DO
Clegg
A
A
Villa
J
J
Meldolesi
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002155:Calsequestrin / Q000502:physiology*; D002454:Cell Differentiation; D002455:Cell Division; D002460:Cell Line; D002645:Chickens; D006706:Homeostasis; D008854:Microscopy, Electron; D024510:Muscle Development; D009132:Muscles / Q000378:metabolism* / Q000648:ultrastructure; D051381:Rats
D002155:Calsequestrin; D002118:Calcium