A study of the mechanisms of excitation-contraction coupling in frog skeletal muscle based on measurements of [Ca2+] transients inside the sarcoplasmic reticulum

Abstract

[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.

Keywords: Ca signaling; Excitation–contraction coupling; Ryanodine receptor; Sarcoplasmic reticulum.