J. Gen. Physiol., Volume 111, Number 4, April 1, 1998 505-519

Steady State Relation between Cytoplasmic Free Ca²⁺ Concentration and Force in Intact Frog Skeletal Muscle Fibers

Masato Konishi, and Masaru Watanabe

From the Department of Physiology, The Jikei University School of Medicine, Tokyo 105, Japan

The steady state relation between cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) and force was studied in intact skeletal muscle fibers of frogs. Intact twitch fibers were injected with the dextran-conjugated Ca²⁺ indicator, fura dextran, and the fluorescence signals of fura dextran were converted to [Ca²⁺]_i using calibration parameters previously estimated in permeabilized muscle fibers (Konishi and Watanabe. 1995. J. Gen. Physiol. 106:1123-1150). In the first series of experiments, [Ca²⁺]_i and isometric force were simultaneously measured during high K⁺ depolarization. Slow changes in [Ca²⁺]_i and force induced by 15-30 mM K⁺ appeared to be in equilibrium, as instantaneous [Ca²⁺]_i versus force plot tracked the common path in the rising and relaxation phases of K⁺ contractures. In the second series of experiments, 2,5-di-tert-butylhydroquinone (TBQ), an inhibitor of the sarcoplasmic reticulum Ca²⁺ pump, was used to decrease the rate of decline of [Ca²⁺]_i after tetanic stimulation. The decay time courses of both [Ca²⁺]_i and force were dose-dependently slowed by TBQ up to 5 µM; the instantaneous [Ca²⁺]_i- force relations were nearly identical at 1 µM TBQ, suggesting that the change in [Ca²⁺]_i was slow enough to reach equilibrium with force. The [Ca²⁺]_i-force data obtained from the two types of experiments were consistent with the Hill curve using a Hill coefficient of 3.2-3.9 and [Ca²⁺]_i for half activation (Ca₅₀) of 1.5-1.7 µM. However, if fura dextran reacts with Ca²⁺ with a 2.5-fold greater K_d as previously estimated from the kinetic fitting (Konishi and Watanabe. 1995. J. Gen. Physiol. 106:1123-1150), Ca₅₀ would be 3.7-4.2 µM. We also studied the [Ca²⁺]-force relation in skinned fibers under similar experimental conditions. The average Hill coefficient and Ca₅₀ were estimated to be 3.3 and 1.8 µM, respectively. Although uncertainties remain about the precise levels of [Ca²⁺]_i, we conclude that the steady state force is a 3rd to 4th power function of [Ca²⁺]_i, and Ca₅₀ is in the low micromolar range in intact frog muscle fibers, which is in reasonable agreement with results obtained from skinned fibers.

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