A Mechanism for Both Capacitative Ca 2+ Entry and Excitation-Contraction Coupled Ca 2+ Release by the Sarcoplasmic Reticulum of Skeletal Muscle Cells

We have previously established that L6 skeletal muscle cell cultures display capacitative calcium entry (CCE), a phenomenon established with other cells in which Ca 2+ uptake from outside cells increases when the endoplasmic reticulum (sarcoplasmic reticulum in muscle, or SR) store is decreased. Evidence for CCE rested on the use of thapsigargin (Tg), an inhibitor of the SR CaATPase and consequently transport of Ca 2+ from cytosol to SR, and measurements of cytosolic Ca 2+ . When Ca 2+ is added to Ca 2+ -free cells in the presence of Tg, the measured cytosolic Ca 2+ rises. This has been universally interpreted to mean that as SR Ca 2+ is depleted, exogenous Ca 2+ crosses the plasma membrane, but accumulates in the cytosol due to CaATPase inhibition. Our goal in the present study was to examine CCE in more detail by measuring Ca 2+ in both the SR lumen and the cytosol using established fluorescent dye techniques for both. Surprisingly, direct measurement of SR Ca 2+ in the presence of Tg showed an increase in luminal Ca 2+ concentration in response to added exogenous Ca 2+ . While we were able to reproduce the conventional demonstration of CCE—an increase of Ca 2+ in the cytosol in the presence of thapsigargin—we found that this process was inhibited by the prior addition of ryanodine (Ry), which inhibits the SR Ca 2+ release channel, the ryanodine receptor (RyR). This was also unexpected if Ca 2+ enters the cytosol first. When Ca 2+ was added prior to Ry, the later was unable to exert any inhibition. This implies a competitive interaction between Ca 2+ and Ry at the RyR. In addition, we found a further paradox: we had previously found Ry to be an uncompetitive inhibitor of Ca 2+ transport through the RyR during excitation-contraction coupling. We also found here that high concentrations of Ca 2+ inhibited its own uptake, a known feature of the RyR. We confirmed that Ca 2+ enters the cells through the dihydropyridine receptor (DHPR, also known as the L-channel) by demonstrating inhibition by diltiazem. A previous suggestion to the contrary had used Mn 2+ in place of direct Ca 2+ measurements; we showed that Mn 2+ was not inhibited by diltiazem and was not capacitative, and thus not an appropriate probe of Ca 2+ flow in muscle cells. Our findings are entirely explained by a new model whereby Ca 2+ enters the SR from the extracellular space directly through a combined channel formed from the DHPR and the RyR. These are known to be in close proximity in skeletal muscle. Ca 2+ subsequently appears in the cytosol by egress through a separate, unoccupied RyR, explaining Ry inhibition. We suggest that upon excitation, the DHPR, in response to the electrical field of the plasma membrane, shifts to an erstwhile-unoccupied receptor, and Ca 2+ is released from the now open RyR to trigger contraction. We discuss how this model also resolves existing paradoxes in the literature, and its implications for other cell types.