Since the discovery of the ATP-dependent Ca2+ transport by SR a little over two decades ago, remarkable progress has been made in understanding the kinetic mechanism of Ca2+ transport and ATP hydrolysis and the role of phosphorylated enzyme intermediates in the energetics of active ion transport. Significant information has accumulated on the structure and composition of the SR membrane, on the primary amino acid sequence of the Ca2+-pump protein, and on the adaptive changes in the Ca2+-transport function during embryonic development and muscle activity. The discovery of the charge movement as a step in EC coupling and the use of novel optical probes for analyzing potential and calcium transients in living muscle changed the enigma of EC coupling into a well-defined problem that is clearly open to rational solutions. Studies on the structure, composition, and function of the isolated components of the T-SR system have just begun. The effectiveness of this approach will depend on successful maintenance of the functionally intact structure of the T-SR junction during the disruption of the muscle, which is required for the isolation of pure membrane elements. Reconstitution of a functionally competent junctional complex from isolated components is the ultimate aim of these studies, but the path toward that goal is so difficult that much of the mechanism of EC coupling may be solved by electrophysiologists, before reconstitution is achieved. The avalanche of information on Ca2+ releases induced by various agents under diverse and sometimes ill-defined conditions led to formulation of a series of hypothetical mechanisms. Of these, Ca2+-induced Ca2+ release promises to be an important element of the physiological Ca2+-release process, but few of the other proposed mechanisms can be eliminated from consideration at this stage. The impressive progress of the last few years has left several fundamental problems largely unsolved. Among these are the physical mode of translocation of Ca2+ across the membrane and the molecular mechanism of the coupling of Ca2+ transport to ATP hydrolysis; the regulation of the concentration of the Ca2+-pump protein and calcium in the SR of fast and slow skeletal, cardiac, and smooth muscles; the gating mechanisms that regulate the graded release of calcium from the SR and the composition and biochemical characterization of the triad; the role of SR membrane potential in the regulation of Ca2+ fluxes in vivo; the permeability of SR membranes in living muscle; the functional significance of protein-protein interactions in the SR with respect to Ca2+ transport and permeability control.(ABSTRACT TRUNCATED AT 400 WORDS)

The purified ATPase of sarcoplasmic reticulum was covalently labeled with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-IAEDANS) or with iodoacetamidofluorescein (IAF). In reconstituted vesicles containing both types of ATPase molecules fluorescence energy transfer was observed from the IAEDANS (donor) to the IAF (acceptor) fluorophore as determined by the ratio of donor and acceptor fluorescence intensities, and by nanosecond decay measurements of donor fluorescence in the presence or absence of the acceptor. The observed energy transfer may arise by random collisions between ATPase molecules due to Brownian motion or by formation of complexes containing several ATPase molecules. Experimental distinction between these two models of energy transfer is possible based on predictions derived from mathematical models. Up to tenfold dilution of the lipid phase of reconstituted vesicles with egg lecithin had no measurable effect upon the energy transfer, suggesting that random collision between ATPase molecules in the lipid phase is not the principal cause of the observed effect. Addition of unlabeled ATPase in five- to tenfold molar excess over the labeled molecules abolished energy transfer. These observations together with electron microscopic and chemical cross-linking studies support the existence of ATPase oligomers in the membrane with sufficiently long lifetimes for energy transfer to occur. A hypothetical equilibrium between monomeric and tetrameric forms of the ATPase governed by the membrane potential is proposed as the structural basis of the regulation of Ca uptake and release by sarcoplasmic reticulum membranes during muscle contraction and relaxation.

Fragmented sarcoplasmic reticulum (FSR) membranes isolated from rabbit skeletal muscle are impermeable to inulin-' 4 C (mol wt 5,000), and dextran-' 4 C (mol wt 15,000-90,000) at pH 7.0-9.0, yielding an excluded space of 4-5 ul/mg microsomal protein. In the same pH range urea and sucrose readily penetrate the FSR membrane. EDTA or EGTA (1 m) increased the permeability of microsomes to inulin-14C or dextran-' 4 C at pH 8-9, parallel with the lowering of the FSR-bound Ca + + content from initial levels of 20 nmoles/mg protein to 1-3 nmoles/mg protein. EGTA was as effective as EDTA, although causing little change in the Mg++ content of FSR. The permeability increase caused by chelating agents results from the combined effects of high pH and cation depletion. As inulin began to penetrate the membrane there was an abrupt fall in the rate of Ca + + uptake and a simultaneous rise in ATPase activity. At 40°C inulin penetration occurred at pH 7.0 with I m. EDTA and at pH 9.0 without EDTA, suggesting increased permeability of FSR membranes. This accords with the higher rate of Ca++ release from FSR at temperatures over 30 0 C. The penetration of microsomal membranes by anions is markedly influenced by charge effects. At low ionic strength and alkaline pH acetate and C1 are partially excluded from microsomes when applied in concentrations not exceeding I mM, presumably due to the Donnan effect. Penetration of microsomal water space by acetate and Cl occurs at ionic strengths sufficiently high to minimize charge repulsions.

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