a study of the mechanisms of excitation–contraction coupling in frog skeletal muscle based on measurements of [ca2+] 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.