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METHODS IN CELL PHYSIOLOGY

Cross-bridge blocker BTS permits direct measurement of SR Ca²⁺ pump ATP utilization in toadfish swimbladder muscle fibers

Biology Department, University of Pennsylvania, Philadelphia, Pennsylvania 19104; and Marine Biological Laboratory, Woods Hole, Massachusetts 02543

Submitted 15 January 2003 ; accepted in final form 22 May 2003

Because the major processes involved in muscle contraction require rapid utilization of ATP, measurement of ATP utilization can provide important insights into the mechanisms of contraction. It is necessary, however, to differentiate between the contribution made by cross-bridges and that of the sarcoplasmic reticulum (SR) Ca²⁺ pumps. Specific and potent SR Ca²⁺ pump blockers have been used in skinned fibers to permit direct measurement of cross-bridge ATP utilization. Up to now, there was no analogous cross-bridge blocker. Recently, N-benzyl-p-toluene sulfonamide (BTS) was found to suppress force generation at micromolar concentrations. We tested whether BTS could be used to block cross-bridge ATP utilization, thereby permitting direct measurement of SR Ca²⁺ pump ATP utilization in saponin-skinned fibers. At 25 µM, BTS virtually eliminates force and cross-bridge ATP utilization (both <4% of control value). By taking advantage of the toadfish swimbladder muscle's unique right shift in its force-Ca²⁺ concentration ([Ca²⁺]) relationship, we measured SR Ca²⁺ pump ATP utilization in the presence and absence of BTS. At 25 µM, BTS had no effect on SR pump ATP utilization. Hence, we used BTS to make some of the first direct measurements of ATP utilization of intact SR over a physiological range of [Ca²⁺]at 15°C. Curve fits to SR Ca²⁺ pump ATP utilization vs. pCa indicate that they have much lower Hill coefficients (1.49) than that describing cross-bridge force generation vs. pCa (5). Furthermore, we found that BTS also effectively eliminates force generation in bundles of intact swimbladder muscle, suggesting that it will be an important tool for studying integrated SR function during normal motor behavior.

muscle energetics; skinned muscle fibers; sarcoplasmic reticulum calcium ion pumps; cross bridges

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