DIRECT MONITORING OF THE CALCIUM-CONCENTRATION IN THE SARCOPLASMIC AND ENDOPLASMIC-RETICULUM OF SKELETAL-MUSCLE MYOTUBES

Direct monitoring of the free Ca2+ concentration in the sarcoplasmic r eticulum (SR) was carried out in rat skeletal myotubes transfected wit h a specifically targeted aequorin chimera (srAEQ). Myotubes were also transfected with a chimeric aequorin (erAEQ) that we have demonstrate d previously is retained in the endoplasmic reticulum (ER). Immunoloca lization analysis showed that although both recombinant proteins are d istributed in an endomembrane network identifiable with immature SR, t he erAEQ protein was retained also in the perinuclear membrane. The di fficulty of measuring [Ca2+] in 100-1000 mu M range was overcome with the use of the synthetic coelenterazine analogue, coelenterazine n. We demonstrate that the steady state levels of [Ca2+] measured with srAE Q is around 300 mu M, whereas that measured with erAEQ is significantl y lower, i.e. around 200 mu M. The effects of caffeine, high KCl, and nicotinic receptor stimulation, in the presence or absence of external calcium or after blockade of the Ca-ATPase, were investigated with bo th chimeras. The kinetics of [Ca2+] changes revealed by the erAEQ were similar, but not identical, neither quantitatively nor qualitatively, to those monitored with the srAEQ, indicating that at this stage of m uscle development, differences exist between SR and ER in their mechan isms of Ca2+ handling. The functional implications of these findings a re discussed.