Ca 2 + Release from the Sarcoplasmic Reticulum Compared in Amphibian and Mammalian Skeletal Muscle

Puzzled by recent reports of differences in specific ligand binding to muscle Ca 2+ channels, we quantitatively compared the flux of Ca 2+ release from tile sarcoplasmic reticulum (SR) in skeletal muscle fibers of an amphibian (frog) and a mammal (rat), voltage clamped in a double Vaseline gap chamber. The determinations of release flux were carried out by the "removal" method and by measuring the rate of Ca 2+ binding to dyes in large excess over other Ca 2+ buffers. To have a more meaningful comparison, the effects of stretching the fibers, of rapid changes in temperature, and of changes in the Ca 2§ content of the SR were studied in both species. In both frogs and rats, the release flux had an early peak followed by fast relaxation to a lower sustained release. The peak and steady values of release flux, Rp and R~, were influenced little by stretching. Rp in frogs was 31 mM/s (SEM = 4, n = 24) and in rats 7 42 mM/s (n = 12). R~ was 9 + 1 and 3 40.7 mM/s in frogs and rats, respectively. Transverse (T) tubule area, estimated from capacitance measurements and norrealized to fiber volume, was greater in rats (0.61 + 0.04 ixm -1) than in frogs (0.48 40.04 ixm-l), as expected from the greater density of T tubuli. Total Ca in the SR was estimated as 3.4 --0.6 and 1.9 + 0.3 mmol/ l i ter myoplasmic water in frogs and rats. With the above figures, the steady release flux per unit area of T tubule was found to be fourfold greater in the frog, and the steady permeability of the junctional SR was about threefold greater. The ratio Rp/Rs was ~ 2 in rats at all voltages, whereas it was greater and steeply voltage dependent in frogs, going through a maximum of 6 at 4 0 mV, then decaying to ~3.5 at high voltage. Both Rp and R~ depended strongly on the temperature, but their ratio, and its voltage dependence, did not. Assuming that the peak of Ca 2+ release is contributed by release channels not in contact with voltage sensors, or not under their direct control, the greater ratio in frogs may correspond to the relative excess of Ca 2+ release channels over voltage sensors apparent in binding measurements. From the marked differences in voltage dependence of the ratio, as well as consideration of Ca2+-induced release models, we derive indications of fundamental differences in control mechanisms between mammalian and amphibian muscle. I N T R O D U C T I O N The flux of Ca 2+ release f rom the sarcoplasmic reticulure, measured in skeletal muscle fibers unde r voltage clamp, includes two well-defined kinetic components , a fast early peak and a mainta ined phase of release (BayAddress correspondence to Dr. E. R/os, Department of Molecular Biophysics and Physiology, Rush University School of Medicine, 1750 West Harrison Street, Chicago, IL 60612. Dr. Shirokova's permanent address is A. A. Bogomoletz Institute of Physiology, Ukrainian National Academy of Sciences, Bogomoletz St.4, 252601 GSP, Kiev 24, Ukraine. Dr. Garcfa's current address is Department of Physiology, Colorado State University, Fort Collins, CO 80523. Dr. Pizarro's permanent address is Departamento de Biotisica, Facultad de Medicina, Montevideo, Uruguay. lor et al., 1983; Melzer et al., 1984, 1987). It has been proposed that these componen t s reflect release through two different sets of channels (Rios and Pizarro, 1988), one activated by Ca 2+ and the o ther directly by voltage. This proposal is consistent with experimental results o f J a c q u e m o n d et al. (1991) using intrac e l l u l a r C a 2+ buffers, and the "skipping" pairing of release channels with T m e m b r a n e tetrads (putative DHP receptors /vol tage sensors) found in the triadic junction of fish (Block et al., 1988) and per ipheral couplings of embryonic mouse, frog slow, and cultured human muscle (Franzini-Armstrong and Kish, 1995). In this proposal, release channels not paired with tetrads are Ca 2+ opera ted and contr ibute the peak c o m p o n e n t of release, whereas those paired with DHP recep to r s / 1 j. GEN. PHYSIOL. 9 The Rockefeller University Press ~ 0022-1295/96/01/1/18 $2.00 Volume 107 January 1996 1-18 on Jne 0, 2017 D ow nladed fom Published January 1, 1996