Interaction of phosphatidic acid and phosphatidylserine with the Ca2+-ATPase of sarcoplasmic reticulum and the mechanism of inhibition

Abstract

The sarcoplasmic reticulum of skeletal muscle contains anionic phospholipids as well as the zwitterionic phosphatidylcholine and phosphatidylethanolamine. Here we study the effects of anionic phospholipids on the activity of the Ca2+-ATPase purified from the membrane. Reconstitution of the Ca2+-ATPase into dioleoylphosphatidylserine [di(C18:1)PS] or dioleoylphosphatidic acid [di(C18:1)PA] leads to a decrease in ATPase activity. Measurements of the quenching of the tryptophan fluorescence of the ATPase by brominated phospholipids give a relative binding constant for the anionic lipids compared with dioleoylphosphatidylcholine close to 1 and suggest that phosphatidic acid only binds to the ATPase at the bulk lipid sites around the ATPase. Addition of di(C18:1)PS or di(C18:1)PA to the ATPase in the short-chain dimyristoleoylphosphatidylcholine [di(C14:1)PC] reverse the effects of the short-chain lipid on ATPase activity and on Ca2+ binding, as revealed by the response of tryptophan fluorescence intensity to Ca2+ binding. It is concluded that the lipid headgroup and lipid fatty acyl chains have separate effects on the function of the ATPase. The anionic phospholipids have no significant effect on Ca2+ binding to the ATPase; the level of Ca2+ binding to the ATPase, the affinity of binding and the rate of dissociation of Ca2+ are unchanged by reconstitution into di(C18:1)PA. The major effect of the anionic lipids is a reduction in the maximal level of binding of MgATP. This is attributed to the formation of oligomers of the Ca2+-ATPase, in which only one molecule of the ATPase can bind MgATP dimers in di(C18:1)PS and trimers or tetramers in di(C18:1)PA. The rates of phosphorylation and dephosphorylation for the proportion of the ATPase still able to bind ATP are unaffected by reconstitution. Larger changes were observed in the level of phosphorylation of the ATPase by Pi, which became very low in the anionic phospholipids. The fluorescence response to Mg2+ for the ATPase labelled with 4-(bromomethyl)-6,7-dimethoxycoumarin was also changed in di(C18:1)PS and di(C18:1)PA, so that effects of Mg2+ became comparable with those seen on phosphorylation for the unreconstituted ATPase. The anionic phospholipids could induce a conformational change in the ATPase on binding Mg2+ equivalent to that normally induced by phosphorylation or by binding inhibitors such as thapsigargin.

Anionic phospholipids decrease the rate of slippage on the Ca2+-ATPase of sarcoplasmic reticulum

“…The rate of hydrolysis of ATP was not significantly changed by the presence of 10 mol % anionic phospholipid (Table 1). This is consistent with measurements of ATPase activity for the ATPase reconstituted into membrane fragments, where ATPase activity was unaffected by the presence of 10 mol % DOPA or DOPS and decreases at higher mole fractions of anionic phospholipid [23].…”

“…Light SR vesicles were prepared from rabbit skeletal muscle as described by Dalton et al [23] ; the vesicles contain predominantly Ca# + -ATPase with some calsequestrin and 53 kDa glycoprotein [24], the latter two being soluble proteins that are lost during the reconstitution process. Reconstituted vesicles were prepared by a modification of the method described by Levy et al [25].…”

“…the presence of phosphatidylinositol, phosphatidylserine or phosphatidic acid at low mole fractions has no effect on activity, and at higher mole fractions leads to inhibition [23]. The native SR membrane contains about 2 mol % phosphatidylserine and 8 mol % of PtdIns and PtdIns(4)P, but no PtdIns(4,5)P # [29,30].…”

“…The rate constants for the E2 E1 change were calculated from the parameters described in Henderson et al [32]. The affinity for ATP [33], the rate of phosphorylation [23] and the rate of dephosphorylation [34] of the phosphorylated intermediate E2P have been set at the experimentally determined values. Ca# + binding has been simplified to a two-step binding process, since this was found to give the same result as the full binding model described by Henderson et al [32].…”

Deletions in the Acidic Lipid-binding Region of the Plasma Membrane Ca2+ Pump

“…6). Under these conditions, increasing concentrations of PA stimulated the activity of the recombinant wild type reaching a maximum value of about 200% at 30 M PA, and then it decreased at higher concentrations of PA, probably reflecting the inhibitory effect of acidic lipids reported previously (8,13,27). In contrast, PA minimally affected the activity of mutant d296 -349 increasing its activity up to a maximum of about 120% at 75 M PA.…”