Effect of Mg2+ concentration on Ca2+ uptake kinetics and structure of the sarcoplasmic reticulum membrane

Abstract

Direct measurements of phosphorylation of the Ca2+ ATPase of the sarcoplasmic reticulum (SR) have shown that the lifetime of the first phosphorylated intermediate in the Ca2+ transport cycle, E1 approximately P, increases with decreasing [Mg2+] (Dupont, Y. 1980. Eur. J. Biochem. 109:231-238). Previous x-ray diffraction work (Pascolini, D., and J.K. Blasie. 1988. Biophys. J. 54:669-678) under high [Mg2+] conditions (25 mM) indicated that changes in the profile structure of the SR membrane could be responsible for the low-temperature transient trapping of E1 approximately P that occurs at temperatures below 2-3 degrees C, the upper characteristic temperature th for lipid lateral phase separation in the membrane. We now present results of our study of the Ca2+ uptake kinetics and of the structure of the SR membrane at low [Mg2+] (less than or equal to 100 microM). Our results show a slowing in the kinetics of both phases of the Ca2+ uptake process and an increase in the duration of the plateau of the fast phase before the onset of the slow phase, indicating an increase in the lifetime (transient trapping) of E1 approximately P. Calcium uptake kinetics at low [Mg2+] and moderately low temperature (approximately 0 degree C) are similar to those observed at much lower temperatures (approximately -10 degrees C) at high [Mg2+]. The temperature-induced structural changes that we observed at low [Mg2+] are much more pronounced than those found to occur at higher [Mg2+]. Also, at the lower [Mg2+] the upper characteristic temperature th for lipid lateral phase separation was found to be higher, at approximately 8-10 degrees C. Our studies indicate that both temperature and [Mg2+] affect the structure and the functionality (as measured by changes in the kinetics of Ca2+ uptake) of the SR membrane. Membrane lipid phase behavior and changes in the Ca2+ ATPase profile structure seem to be related, and we have found that structural changes are responsible for the slowing of the kinetics of the fast phase of Ca2+ uptake, and could also mediate the effect that [Mg2+] has on E1 approximately P lifetime.

Evidence that lipid lateral phase separation induces functionally significant structural changes in the Ca+2ATPase of the sarcoplasmic reticulum

“…2 (4). Our conclusions concerning the functional relevance of these structural changes observed in the membrane were further supported by our study of the effect of [Mg+21 on the profile structure of the SR membrane, the value of th, and the kinetics of Ca+2 uptake by the SR system (3).…”

“…By studying the effects of several different physicochemical parameters on the lateral phase separation (LPS) behavior of the lipids in the intact SR membrane, we expected to be able to firmly establish the existence of a relation between the behavior of the lipids and the structure and functionality of the Ca+2ATPase, the latter measured by the kinetics of ATP-induced Ca+2 uptake. We had previously studied the kinetics of Ca+2 uptake by both multilayers (relatively low water content) and vesicular dispersions (relatively high water content) of SR membranes (3,4), and found some differences that we attributed to changes in the degree of hydration of the membranes. Our previous studies on the influence of different physico-chemical parameters on the LPS behavior of the lipids in the intact SR membrane (3) did not Step-function electron density models of the SR membrane profile, corresponding to continuous electron density profiles (13 A resolution) calculated directly from the lamellar intensity functions shown in Fig.…”

“…INTRODUCTION Previous x-ray diffraction studies of the sarcoplasmic reticulum (SR)' membrane from our laboratory revealed the existence of a temperature-induced structural transition in the rotationally averaged profile structure of the Ca 2ATPase protein, and established a correlation between the upper characteristic temperature for lateral phase separation (1) of the membrane lipids, th, and the temperature at which the structural transition in the protein was observed (2,3). The temperature-induced changes observed in the lamellar diffraction and corresponding profile structure of the SR membrane at low [Mg+2] are illustrated, for example, in Fig.…”

Lipids and the ocular lens

“…For instance, when the hydrocarbon chains of the lipids are ordered, the rate of calcium pumping by SERCA is lower. This is due to the reduced rate of phosphorylation at the catalytic site of the enzyme (86)(87)(88)(89). SERCA pump function is also infl uenced by bilayer thickness ( 90,91 ), phospholipid composition ( 92 ), fl uidity ( 88,89,93,94 ), and hydrocarbon-chain saturation ( 94,95 ).…”

Effect of Ca2+ binding on the profile structure of the sarcoplasmic reticulum membrane using time-resolved x-ray diffraction

“…"Large-scale" (i.e., long-range) structural changes in the Ca2+-ATPase occurring during specific steps in the Ca2+ transport cycle have been directly investigated via time-resolved x-ray diffraction from partially dehydrated, oriented SR membrane multilayers (5)(6)(7)(8)(9). Such time-resolved experiments have used the flash-photolysis of the ultraviolet (UV)-sensitive molecule caged-ATP (and caged-ADP as an essential control) to initiate enzyme phosphorylation rapidly and synchronously to ' Abbreviation used in this paper: SR, sarcoplasmic reticulum.…”