A new approach for measuring real-time calcium pumping and SR function in muscle fibers.

Abstract

Different muscles are designed to perform a wide variety of motor activities that extend over a large range of frequencies. To increase our understanding of the molecular basis for these different designs, we are attempting to model the activation-relaxation cycle of different muscles by obtaining kinetic information about each of the constituent processes (1, 2). An important component in muscle relaxation is Ca*+ sequestration by sarcoplasmic reticulum (SR)-Ca*’ pumps, but the pump turnover rate in different fiber types is not known precisely. Previous measurements of SR function have generally involved one of two approaches. One approach involves homogenizing muscle, making SR preparations and measuring the rate of Ca*+ accumulation in the presence of oxalate (e.g., 3). It is unclear, however, whether homogenization alters the pumping rate compared to that of intact SR in muscle fibers. Further, the presence of oxalate probably increases the rate of accumulation above that in normal fibers by reducing the free Ca*+ concentration inside the SR. An alternate approach involves physiological measurements of intracellular free [Ca*+] using calcium sensitive dyes (e.g., 1, 4). However, this approach provides direct information only about the small amount of Ca*+ that is actually free in the myoplasm. The total movements of Ca*+ can only be estimated by modelling the large quantities of other Ca*+ buffers in the cell (e.g., parvalbumin and troponin; 4), but the amounts and the kinetics of these calcium buffers are not always known in detail. We have developed a technique and an instrument that permit direct measurements of Ca*+ sequestration by intact SR of muscle fibers as a function of [Ca2+rree]. Single muscle fibers are exposed to 50 wg/ml saponin (20 min), which permeablizes the cell membrane, but leaves the SR membrane intact (5). The “skinned” muscle fibers are then bathed in solutions that contain various concentrations of Ca2+rree; and calcium sequestration (i.e., Ca *+ leaving the bath) is monitored by using a Ca*+ sensitive dye, FURA-2. This approach solves two important problems. First, during the “skinning” procedure, most of the intracellular Ca*+-buffer (the soluble parvalbumin) leaks out of the cell membrane and is washed away. Second, the amounts of remaining intracellular buffers (e.g., troponin), become very small compared to the total buffer in the bath, which is well defined in terms of amounts and kinetics, and can be accurately calibrated. Hence Ca*+ sequestration by the SR can be accurately measured as a removal of Ca*+ from the bathing solution.