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

Abstract

The Ca2+-ATPase is an integral transmembrane Ca2+ pump of the sarcoplasmic reticulum (SR). Crystallization of the cytoplasmic surface ATPase molecules of isolated scallop SR vesicles was studied at various calcium concentrations by negative stain electron microscopy. In the absence of ATP, round SR vesicles displaying an assembly of small crystalline patches of ATPase molecules were observed at 18 µM [Ca2+]. These partly transformed into tightly elongated vesicles containing ATPase crystalline arrays at low [Ca2+] (≤1.3 µM). The arrays were classified as ‘’tetramer’’, “two-rail” (like a railroad) and ‘’monomer’’. Their crystallinity was low, and they were unstable. In the presence of ATP (5 mM) at a low [Ca2+] of ~0.002 µM, “two-rail” arrays of high crystallinity appeared more frequently in the tightly elongated vesicles and the distinct tetramer arrays disappeared. During prolonged (~2.5 h) incubation, ATP was consumed and tetramer arrays reappeared. A specific ATPase inhibitor, thapsigargin, prevented both crystal formation and vesicle elongation in the presence of ATP. Together with the second part of this study, these data suggest that the ATPase forms tetramer units and longer tetramer crystalline arrays to elongate SR vesicles, and that the arrays transform into more stable “two-rail” forms in the presence of ATP at low [Ca2+].

Ca2+ Dependent Formation/Collapse of Cylindrical Ca2+-ATPase Crystals in Scallop Sarcoplasmic Reticulum (SR) Vesicles: A Possible Dynamic Role of SR in Regulation of Muscle Contraction

“…To overcome this, a stable SR vesicle preparation with a high calcium transport activity was isolated from scallop [ 2 ]. As reported in the previous paper of the scallop SR vesicles [ 3 ], the data obtained using this preparation confirm the elongation of vesicles by calcium-sensitive ATPase crystallization previously observed using rabbit SR vesicles. Moreover, an ATPase tetramer was recognized as a structural unit of the crystalline arrays, and ATP was shown to stabilize them.…”

“…Table 1 and Figure 1 summarize the ATPase particle (~40 Å) dispositions (see Ref. [ 3 ] for details) observed in SR vesicles and the vesicular shapes at different calcium concentrations (approx. 0.002–59 µM Ca 2+ ) in the presence of 5 mM ATP (see Supplementary Table S1 for full details).…”

“…The crystalline arrays of ATPase molecules were subclassified into two types: “two-rail” (like a railroad track; see Figure 3c,c’ in Ref. [ 3 ]) and monomer (like a lattice of monomers; see Figure 3d,d’ in Ref. [ 3 ]).…”

“…[ 3 ]) and monomer (like a lattice of monomers; see Figure 3d,d’ in Ref. [ 3 ]). The non-crystalline arrays contained an assembly of various types of small ATPase crystals, including fragmented-ladder and/or “Lego” types, i.e., what we call a crystal patch assembly (mosaic crystallization; see Figure 5a in Ref.…”

“…The scallop SR directly associates with the plasma membrane via junctional feet-like structures of ryanodine receptors (RyR; the calcium-release channel of SR) [ 6 , 7 ] (see Figure 1 in Ref. [ 3 ]). By contrast, the SR of vertebrate cross-striated skeletal muscle associates with T tubules via the junctional feet-like structures of RyRs [ 8 ].…”