Effects of intracellular ruthenium red on excitation‐contraction coupling in intact frog skeletal muscle fibres.

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Abstract
1. Ruthenium Red (RR) blocks sarcoplasmic reticulum (SR) Ca2+ release in disrupted muscle preparations and this block has been used as a marker for the physiological Ca2+ release pathway. To investigate whether RR can also affect SR Ca2+ release in living muscle, optical signals reflecting Ca2+ release have been measured in intact single frog twitch fibres microinjected with RR. 2. The total myoplasmic concentration of RR, [RRT], was obtained from measurements of RR-related absorbance and apparent diffusion constant, Dapp, of RR in myoplasm was estimated. The value of Dapp was about 1/30 of that expected and can be explained if the majority of RR (approximately 97%) was bound in myoplasm and free [RR] was only 1/30 of [RRT]. 3. Sarcoplasmic reticulum Ca2+ release following action potential stimulation was assessed from a Ca2+-related change in intrinsic birefringence. The birefringence signal was blocked in the presence of RR and the degree of block was clearly dependent upon [RRT]. At 16 degrees C the estimated [RRT] for half-block of the birefringence signal was 23 +/- 4 microM (+/- S.E. of mean; n = 3), and for half-block of the Ca2+ release process itself was 72 +/- 14 microM. The estimated free [RR] for half-block is then 0.8 +/- 0.1 and 2.4 +/- 0.5 microM, respectively. In the cold (6-8 degrees C), the half-blocking concentration of RR, referred to [RRT], appeared to be about 3-fold smaller than that observed at 16 degrees C. 4. The values estimated for the free [RR] which caused half-block of Ca2+ release in intact muscle fibres are in the range reported for RR's action in disrupted preparations, thus supporting the conclusion that the RR-blockable channel observed in disrupted muscle is the physiologically important Ca2+ release channel. 5. Intramembrane charge movements in skeletal muscle are thought to underlie the dependence of SR Ca2+ release on transverse tubular membrane potential. Charge movements were measured in RR-injected fibres at 4-6 degrees C using a three-microelectrode, middle-of-the-fibre voltage-clamp technique. Injected fibres did not survive well in solutions made hypertonic to prevent fibre movement and allow measurement of suprathreshold charge; therefore charge movements below contraction threshold were studied in isotonic solution.(ABSTRACT TRUNCATED AT 400 WORDS)