A Mechanism for Both Capacitative Ca²⁺Entry and Excitation-Contraction Coupled Ca²⁺Release by the Sarcoplasmic Reticulum of Skeletal Muscle Cells

Abstract

We have previously established that L6 skeletal muscle cell cultures display capacitative calcium entry (CCE), a phenomenon established with other cells in which Ca2+ 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 Ca2+ from cytosol to SR, and measurements of cytosolic Ca2+. When Ca2+ is added to Ca2+-free cells in the presence of Tg, the measured cytosolic Ca2+ rises. This has been universally interpreted to mean that as SR Ca2+ is depleted, exogenous Ca2+ 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 Ca2+ in both the SR lumen and the cytosol using established fluorescent dye techniques for both. Surprisingly, direct measurement of SR Ca2+ in the presence of Tg showed an increase in luminal Ca2+ concentration in response to added exogenous Ca2+. While we were able to reproduce the conventional demonstration of CCE—an increase of Ca2+ 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 Ca2+ release channel, the ryanodine receptor (RyR). This was also unexpected if Ca2+ enters the cytosol first. When Ca2+ was added prior to Ry, the later was unable to exert any inhibition. This implies a competitive interaction between Ca2+ and Ry at the RyR. In addition, we found a further paradox: we had previously found Ry to be an uncompetitive inhibitor of Ca2+ transport through the RyR during excitation-contraction coupling. We also found here that high concentrations of Ca2+ inhibited its own uptake, a known feature of the RyR. We confirmed that Ca2+ 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 Mn2+ in place of direct Ca2+ measurements; we showed that Mn2+ was not inhibited by diltiazem and was not capacitative, and thus not an appropriate probe of Ca2+ flow in muscle cells. Our findings are entirely explained by a new model whereby Ca2+ 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. Ca2+ 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 Ca2+ 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.

A Direct Mass-action Mechanism Explains Capacitative Calcium Entry in Jurkat and Skeletal L6 Muscle Cells

“…1b, ryanodine did not cause inhibition when exogenous Ca 2ϩ was added before Ry. Thus, as we observed previously with L6 muscle cells (14), ryanodine blocked an increase in cytosolic [Ca 2ϩ ], the current hallmark of CCE, but only if added before exogenous Ca 2ϩ . Because ryanodine blocks the ER Ca 2ϩ release channels (the ryanodine receptor, or RyR), the results imply that the origin of cytosolic Ca 2ϩ during CCE may be the ER rather than the extracellular space, which is usually assumed.…”

“…Complementary Results with L6 Cells-We have recently shown that Ca 2ϩ in the SR of the L6 muscle cells increases with increasing exogenous Ca 2ϩ , but the titration appeared qualitatively different from the one we observed in the present study with Jurkat cells; there was considerable inhibition of Ca 2ϩ content as Ca 2ϩ was added at concentrations greater than 3 mM (14). We, therefore, transfected L6 cells with aequorin using methods similar to those used for Jurkat cells and titrated the cells with exogenous Ca 2ϩ .…”

“…Fluorescent Measurements-Measurement of cytosolic Ca 2ϩ was performed as reported previously using Indo PE3 (AM) dye (14). For measurement of the ER Ca 2ϩ of Jurkat cells, the cells were loaded with 8 -10 M concentrations of the dye Mag-Fura in Hanks' balanced salt solution.…”

“…We had previously shown that CCE occurs in L6 skeletal muscle cells (11), and others demonstrated subsequently that CCE occurs in skeletal muscle (12,13). In our prior studies of L6 cells, we determined Ca 2ϩ in both cytosol and ER using fluorescent dyes, finding that Ca 2ϩ enters the ER even in the presence of thapsigargin (14). In the present study, we explored this hypothesis for Jurkat cells, a well studied non-excitable cell line.…”

“…The fluorescence measurements used excitation wavelengths of 347 and 373 nm and emission at 507 nm (14). The ratio of the bound to unbound dye with a dissociation constant of 53 M for Mag-Fura was used to calculate the free ER [Ca 2ϩ ] as described by Grynkiewicz (22).…”