Valinomycin and excitation-contraction coupling in skeletal muscle fibres of the frog

Abstract

1. Experiments were carried out on intact frog skeletal muscle fibres to study the role of H+ and K+ as counter-ions during the release of Ca2+ from the sarcoplasmic reticulum (SR). A specific focus was to test whether valinomycin, a potassium ionophore, markedly reduces or abolishes H+ counter-ions fluxes across the SR membrane in response to electrical stimulation. 2. Single twitch fibres, mounted on an optical bench apparatus and stretch to long sarcomere length (3.6-4.0 microns), were activated by single action potentials (16 degrees C). Two optical signals related to excitation-contraction coupling were measured: (i) the 'second component' of the intrinsic birefringence signal, which is closely related to the myoplasmic free [Ca2+] transient, and (ii) the transient myoplasmic alkalization (delta pH) detectable from the pH indicator Phenol Red, a signal thought to reflect the movement of protons from the myoplasm into the SR in partial electrical exchange for released Ca2+. 3. Exposure of a fibre to 5 microM-valinomycin produced a slight, progressive decrease in the amplitude of the birefringence signal, approximately 5-6% per hour. This result suggests that, if anything, the peak rate at which Ca2+ is released from the sarcoplasmic reticulum is slightly decreased by valinomycin. 4. The amplitude of the Phenol Red delta pH signal, measured after exposure of a fibre to valinomycin for a period of at least 60 min, averaged 0.0020 +/- 0.0002 (+/- S.E.M.); this value is slightly smaller than, but not significantly different from (P greater than 0.05; two-tailed t test) that measured in fibres not exposed to valinomycin (0.0025 +/- 0.0002). This result does not support the idea that valinomycin, but virtue of increasing the flux of K+ into the SR, markedly reduces the flux of protons during Ca2+ release. 5. Our findings of minimal changes in the birefringence and delta pH signals are consistent with the idea that, at the time of Ca2+ release, the potassium conductance of the SR membrane is large and not substantially increased by the addition of valinomycin to Ringer solution.