Ca2+-ATPase Molecules as a Calcium-Sensitive Membrane-Endoskeleton of Sarcoplasmic Reticulum

Abstract

The Ca2+-transport ATPase of sarcoplasmic reticulum (SR) is an integral, transmembrane protein. It sequesters cytoplasmic calcium ions released from SR during muscle contraction, and causes muscle relaxation. Based on negative staining and transmission electron microscopy of SR vesicles isolated from rabbit skeletal muscle, we propose that the ATPase molecules might also be a calcium-sensitive membrane-endoskeleton. Under conditions when the ATPase molecules scarcely transport Ca2+, i.e., in the presence of ATP and ≤ 0.9 nM Ca2+, some of the ATPase particles on the SR vesicle surface gathered to form tetramers. The tetramers crystallized into a cylindrical helical array in some vesicles and probably resulted in the elongated protrusion that extended from some round SRs. As the Ca2+ concentration increased to 0.2 µM, i.e., under conditions when the transporter molecules fully carry out their activities, the ATPase crystal arrays disappeared, but the SR protrusions remained. In the absence of ATP, almost all of the SR vesicles were round and no crystal arrays were evident, independent of the calcium concentration. This suggests that ATP induced crystallization at low Ca2+ concentrations. From the observed morphological changes, the role of the proposed ATPase membrane-endoskeleton is discussed in the context of calcium regulation during muscle contraction.

Elongation and Contraction of Scallop Sarcoplasmic Reticulum (SR): ATP Stabilizes Ca2+-ATPase Crystalline Array Elongation of SR Vesicles

“…They were performed using isolated scallop SR vesicles under the artificial, physiological conditions. The negative staining method employed has limitations because it involves staining with uranyl acetate and drying the sample [ 6 ]; some salt patterns might be formed during drying process and the drying process could itself disturb SR structures. Although these factors might have made the SR classification more complex, we consider the general trends reported to be representative and hope that the theory proposed will stimulate future in vivo study of the SR.…”

“…They will be the subject of a future study. Stabilization of the crystalline arrays by ATP, can be described based on the biological hydrotrope model of ATP [ 24 ], like rabbit Ca 2+ -ATPase crystals [ 6 ], i.e., ATP may return the structure of the scallop Ca 2+ -ATPase molecules into their intrinsic state to help crystallization. The inhibition of ATPase “two-rail” crystallization by TG alone might be specific to scallop, because the formation of two-rail-crystalline arrays of rabbit Ca 2+ -ATPase promoted by decavanadate [ 25 ] (an inhibitor of Ca 2+ -ATPase [ 26 ]) was further enhanced by TG [ 23 , 27 ].…”

“…This lowers the cytoplasmic Ca 2+ concentration, leading to the relaxation of the muscle [ 4 , 5 ]. Recently, SR vesicles isolated from the adult fast-twitch skeletal muscle of rabbit, were observed using negative staining and transmission electron microscopy (TEM) [ 6 ]. At a low Ca 2+ concentration (≤0.9 nM), at which the SR Ca 2+ -ATPase molecules scarcely transport calcium, the surface ATPase molecules (40 Å diameter) formed crystalline arrays with the help of ATP to elongate the SR vesicles.…”

“…However, in this study, the rabbit SR vesicles were too susceptible to cohesion/agglomeration in the presence of ATP to obtain the number of images required for statistical analysis, in particular, to measure the calcium dependencies of ATPase crystallization and vesicular morphology. Thus, the model of a calcium-sensitive membrane endoskeleton proposed for rabbit SR [ 6 ] remained preliminary. On the other hand, earlier the elongated SR vesicles isolated from the cross-striated adductor muscle of scallop were reported to have crystalline arrays of ATPase particles (~40 Å diameter), independent of ATP [ 7 ].…”

“…A scallop SR preparation with a high Ca 2+ -ATPase percentage (~80% of the protein present) was employed in the earlier studies mentioned above, and its calcium transport activity (~4 nmol Ca 2+ /mg of protein/min) [ 7 ] was much lower than the high calcium transport (800–6000 nmol Ca 2+ /mg of protein/min) of rabbit skeletal muscle SR [ 14 , 15 ]. In the present work, we used the isolated scallop SR with a high calcium transport activity (~1000 nmol Ca 2+ /mg of protein/min) but with a low ATPase content [ 15 ], to find out whether the calcium dependence of crystalline array formation and elongation of the SR observed for rabbit ATPase proteins [ 6 ] is also true for scallop. In the first part of this study, the stability of the scallop ATPase crystalline array was examined in the presence and absence of ATP.…”