Defining the Molecular Components of Calcium Transport Regulation in a Reconstituted Membrane System

Abstract

Using a chemically defined reconstitution system, we performed a systematic study of key factors in the regulation of the Ca-ATPase by phospholamban (PLB). We varied both the lipid/protein and PLB/Ca-ATPase ratios, determined the effects of PLB phosphorylation, and compared the regulatory effects of several PLB mutants, as a function of Ca concentration. The reconstitution system allowed us to determine accurately not only the PLB effects on K(Ca) (Ca concentration at half-maximal activity) of the Ca-ATPase, but also the effects on V(max) (maximal activity). Wild-type PLB (WT-PLB) and two gain-of-function mutants, N27A-PLB and I40A-PLB, showed not only the previously reported increase in K(Ca), but also an increase in V(max). Specifically, V(max) increases linearly with the intramembrane PLB concentration, and is approximately doubled when the sample composition approaches that of cardiac SR. Upon phosphorylation of PLB at Ser-16, the K(Ca) effects were almost completely reversed for WT- and N27A-PLB but were only partially reversed for I40A-PLB. Phosphorylation induced a V(max) increase for WT-PLB, and a V(max) decrease for N27A- and I40A-PLB. We conclude that PLB and PLB phosphorylation affect V(max) as well as K(Ca), and that the magnitude of both effects is sensitive to the PLB concentration in the membrane.

Superinhibitory Phospholamban Mutants Compete with Ca2+ for Binding to SERCA2a by Stabilizing a Unique Nucleotide-dependent Conformational State

“…We also confirmed that PLB molecules of normal inhibitory strength (N30C-PLB) do not significantly affect the V max of the Ca 2ϩ -ATPase (1). This is contrary to conclusions of several recent studies in which PLB was reported to either decrease (22) or increase (20,21,23) the V max of the Ca 2ϩ -ATPase. Using our viral constructs and the 2D12 antibody, Waggoner et al (22) recently noted a modest reduction (ϳ20%) in the V max of SERCA2a co-expressed with wild-type PLB compared with SERCA2a expressed alone.…”

“…4B). The studies in which wild-type PLB and some other PLB mutants were reported to actually increase the V max of the Ca 2ϩ -ATPase were all conducted with the purified rabbit skeletal muscle enzyme co-reconstituted with purified PLB from detergent solution (20,21,23). In this case, it is possible that enzyme protection by PLB during the reconstitution process may have artifactually affected the results, as was recently suggested (23).…”

Lethal Arg9Cys phospholamban mutation hinders Ca 2+ -ATPase regulation and phosphorylation by protein kinase A

“…PLN variants were coreconstituted with purified SERCA (46,47) in lipid bilayer membranes (1,2-dioleoyl-sn-glycero-3-phosphocholine: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPC:DOPE, 4∶1) at molar ratios of 10∶1 PLN:SERCA and 700∶1 lipids:SERCA. The Ca 2þ dependence of the ATPase activity was measured using a coupled enzyme assay at 37°C (48) and monitored as for the PKA-C assays.…”

“…The Ca 2þ dependence of the ATPase activity was measured using a coupled enzyme assay at 37°C (48) and monitored as for the PKA-C assays. Initial rates of SERCA was measured as a function of calcium concentration (pCa), and data were fit to the Hill equation (48).…”

Regulation of the Sarcoplasmic Reticulum Calcium Pump by Divergent Phospholamban Isoforms in Zebrafish

“…Thus, the residue changes at these two positions (Tyr 6 to His and Leu 7 to Met) account for the functional consequences of the zfHEAD chimera. The effect of PLN on the maximal activity of SERCA is only observed at high protein to lipid and PLN to SERCA ratios that mimic cardiac SR membranes (36,47), and this effect is not reversed by PKA-mediated phosphorylation (30). Residues that contribute to the V max stimulation of SERCA are found in the cytoplasmic and transmembrane domains of PLN (30,36,37).…”