Activating and deactivating roles of lipid bilayers on the Ca(2+)-ATPase/phospholamban complex

Abstract

The physicochemical properties of the lipid bilayer shape the structure and topology of membrane proteins and regulate their biological function. Here, we investigated the functional effects of various lipid bilayer compositions on the sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA) in the presence and absence of its endogenous regulator, phospholamban (PLN). In the cardiac muscle, SERCA hydrolyzes one ATP molecule to translocate two Ca(2+) ions into the SR membrane per enzymatic cycle. Unphosphorylated PLN reduces SERCA's affinity for Ca(2+) and affects the enzymatic turnover. We varied bilayer thickness, headgroup, and fluidity and found that both the maximal velocity (V(max)) of the enzyme and its apparent affinity for Ca(2+) (K(Ca)) are strongly affected. Our results show that (a) SERCA's V(max) has a biphasic dependence on bilayer thickness, reaching maximum activity with 22-carbon lipid chain length, (b) phosphatidylethanolamine (PE) and phosphatidylserine (PS) increase Ca(2+) affinity, and (c) monounsaturated lipids afford higher SERCA V(max) and Ca(2+) affinity than diunsaturated lipids. The presence of PLN removes the activating effect of PE and shifts SERCA's activity profile, with a maximal activity reached in bilayers with 20-carbon lipid chain length. Our results in synthetic lipid systems compare well with those carried out in native SR lipids. Importantly, we found that specific membrane compositions closely reproduce PLN effects (V(max) and K(Ca)) found in living cells, reconciling an ongoing controversy regarding the regulatory role of PLN on SERCA function. Taken with the physiological changes occurring in the SR membrane composition, these studies underscore a possible allosteric role of the lipid bilayers on the SERCA/PLN complex.

FIGURE 1

SERCA activity in bilayers of varying thickness. A, C.) Representative curves of raw and normalized ATPase activity as a function of calcium concentration in 14:1^Δ9-Cis, 16:1^Δ9-Cis, DOPC, 20:1^Δ11-Cis, 22:1^Δ13-Cis and 24:1^Δ15-Cis PC bilayers. Each point represents the average and standard error from three measurements. B.) V_max of SERCA relative to DOPC (V_max/V_max,DOPC) as a function of bilayer chain length. Error bars represent the standard error from at least three separate reconstitutions D.) K_Ca in bilayers of varying thickness. Each bar was determined from the average of ≥ 3 reconstitutions and error bars show the standard error of the mean.

FIGURE 2

SERCA activity in C18 bilayers with different fluidity. A, C.) Raw and normalized ATPase activity in PC bilayers with differences in geometry around the unsaturated bond (18:1^Δ9-trans PC vs DOPC (18:1^Δ9-cis PC)) but identical length of the aliphatic chains. B, D.) ATPase activity in PC bilayers with differences in chain saturation (DOPC vs 18:2^{Δ9, Δ12-cis} PC) but identical length of the aliphatic chains. Error bars correspond to the standard error of three measurements. Averaged K_Ca and relative V_max are shown as insets, with the error bars representing the standard error of three or more separate reconstitutions.

FIGURE 3

Effects of PE and PS head groups on SERCA activity. A,D.) Representative curves of raw and normalized SERCA activity in bilayers composed of DOPC and different mole fractions of 18:1 DOPE as indicated in figure. Error bars represent an average of triplicate measurements and are for some points smaller than the symbol. B.) V_max of SERCA in bilayer with varied amount of PE head group. V_max values are shown relative to the V_max in 0% PE (DOPC). C.) V_max relative to V_max,DOPC in 4:1 (w/w) DOPC:DOPE and 4:1 or 9:1 (w/w) DOPC:DOPS. E.) K_Ca of SERCA in different amounts of PE. ΔK_Ca corresponds to K_{Ca,X% PE} - K_{Ca,0% PE} where X is the mole fraction of PE in the bilayer. F.) K_Ca in DOPC, 4:1 (w/w) DOPC:DOPE and 4:1 or 9:1 (w/w) DOPC:DOPS. Error bars in B,C,E and F represent the standard error of three or more separate reconstitutions. For points where error bars are not visible they are smaller than the symbol.

FIGURE 4

Inhibition of SERCA by AFA-PLN^N27A in bilayers with different head group composition. A,C.) Representative curves of raw and normalized SERCA activity in DOPC and 4:1 (w/w) DOPC:DOPE in the absence and presence of a 10-fold molar excess of AFA-PLN^N27A. Each point shows the average and standard error of three measurements, for some points the error bar is smaller than the symbol. B.) Average V_max in DOPC and 4:1 (w/w) DOPC:DOPE in the presence and absence of AFA-PLN^N27A Values are reported relative to the value in DOPC. D.) Averaged K_Ca values. Error bars in B.) and D.) represent the standard error of three or more separate reconstitutions.

FIGURE 5

Effects of AFA-PLN^N27A on SERCA V_max and K_Ca in bilayers of varying thickness composed of 4:1 (w/w) 14:1 – 20:1^Δ9-Cis PC:DOPE. A, B.) V_max values relative to 4:1 (w/w) DOPC:DOPE in the absence and presence of AFA-PLN^N27A. C.) SERCA Vmax change caused by AFA-PLN^N27A measured as V_{max,+AFA-PLNN27A} / V_{max,-AFA-PLNN27A}, D, E.) K_Ca values in the absence and presence of AFA-PLN^N27A. F.) K_Ca change due caused by AFA-PLN^N27A and measured as ΔK_Ca = K_{Ca,+AFA-PLNN27A} - K_{Ca,-AFA-PLNN27A}. The error bars represent standard error for ≥3 separate reconstitutions.

FIGURE 6

Lipid effects on SERCA and PLN^wt at a physiological lipid concentration. SERCA activity in samples reconstituted with a lipid:PLN^wt:SERCA molar ratio of 150:5:1. A.) Average V_max in the presence and absence of PLN^wt in DOPC and 4:1 (w/w) DOPC:DOPE. V_max is relative to the value in DOPC. B.) Averaged K_Ca values. All error bars represent the standard error of three separate reconstitutions.

FIGURE 7

SERCA and PLN in native lipids. SERCA ATPase activity and AFA-PLN^N27A regulation with proteins reconstituted into lipids extracted from rabbit skeletal muscle SR. A.) Raw activity. B.) Normalized values. Error bars display the standard error of three measurements and are smaller than the symbol for several of the data points.