A study of the mechanisms of excitation-contraction coupling in frog skeletal muscle based on measurements of [Ca 2+ ] transients inside the sarcoplasmic reticulum.

[Ca2+ ] transients inside the sarcoplasmic reticulum (SR) were recorded in frog skeletal muscle twitch fibers under voltage clamp using the low affinity indicator Mag Fluo 4 (loaded in its AM form) with the purpose of studying the effect on Ca2+ release of extrinsic Ca2+ buffers (i.e. BAPTA) added at high concentration to the myoplasm. When the extrinsic Ca2+ buffer is added to the myoplasm, part of the released Ca2+ binds to it, reducing the Ca2+ signal reported by a myoplasmic indicator. This, in turn, hinders the quantification of the amount of Ca2+ released. Monitoring release by measuring [Ca2+ ] inside the SR avoids this problem. The application of extrinsic buffers at high concentration reduced the resting [Ca2+ ] in the SR ([Ca2+ ]SR ) continuously from a starting value close to 400 μM reaching the range of 100 μM in about half an hour. The effect of reducing resting [Ca2+ ]SR on the Ca2+ permeability of the SR activated by voltage clamp depolarization to 0 mV was studied in cells where the myoplasmic [Ca2+ ] ([Ca2+ ]myo ) transients were simultaneously recorded with Rhod2. The Ca2+ release flux was calculated from [Ca2+ ]myo and divided by [Ca2+ ]SR to obtain the permeability. Peak permeability was significantly reduced, from 0.026 ± 0.005 ms-1 at resting [Ca2+ ]SR  = 372 ± 5 μM to 0.021 ± 0.004 ms-1 at resting [Ca2+ ]SR  = 120 ± 16 μM (n = 4, p = 0.03). The time averaged permeability was not significantly changed (0.009 ± 0.003 and 0.010 ± 0.003 ms-1 , at the higher and lower [Ca2+ ]SR respectively). Once the cells were equilibrated with the high buffer intracellular solution, the change in [Ca2+ ]SR (Δ[Ca2+ ]SR ) in response to voltage clamp depolarization (0 mV, 200 ms) in 20 mM BAPTA was significantly lower (Δ[Ca2+ ]SR  = 30.2 ± 3.5 μM from resting [Ca2+ ]SR  = 88.8 ± 13.6 μM, n = 5) than in 40 mM EGTA (Δ[Ca2+ ]SR  = 72.2 ± 10.4 μM from resting [Ca2+ ]SR  = 98.2 ± 15.6 μM, n = 4) suggesting that a Ca2+ activated component of release was suppressed by BAPTA.