The Effects of Mutation on the Regulatory Properties of Phospholamban in Co-Reconstituted Membranes

Abstract

Reconstitution into proteoliposomes is a powerful method for studying calcium transport in a chemically pure membrane environment. By use of this approach, we have studied the regulation of Ca(2+)-ATPase by phospholamban (PLB) as a function of calcium concentration and PLB mutation. Co-reconstitution of PLB and Ca(2+)-ATPase revealed the expected effects of PLB on the apparent calcium affinity of Ca(2+)-ATPase (K(Ca)) and unexpected effects of PLB on maximal activity (V(max)). Wild-type PLB, six loss-of-function mutants (L7A, R9E, I12A, N34A, I38A, L42A), and three gain-of-function mutants (N27A, L37A, and I40A) were evaluated for their effects on K(Ca) and V(max). With the loss-of-function mutants, their ability to shift K(Ca) correlated with their ability to increase V(max). A total loss-of-function mutant, N34A, had no effect on K(Ca) of the calcium pump and produced only a marginal increase in V(max). A near-wild-type mutant, I12A, significantly altered both K(Ca) and V(max) of the calcium pump. With the gain-of-function mutants, their ability to shift K(Ca) did not correlate with their ability to increase V(max). The "super-shifting" mutants N27A, L37A, and I40A produced a large shift in K(Ca) of the calcium pump; however, L37A decreased V(max), while N27A and I40A increased V(max). For wild-type PLB, phosphorylation completely reversed the effect on K(Ca), but had no effect on V(max). We conclude that PLB increases V(max) of Ca(2+)-ATPase, and that the magnitude of this effect is sensitive to mutation. The mutation sensitivity of PLB Asn(34) and Leu(37) identifies a region of the protein that is responsible for this regulatory property.

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).…”

Hydrophobic Imbalance in the Cytoplasmic Domain of Phospholamban Is a Determinant for Lethal Dilated Cardiomyopathy

“…Reconstitution versus Cardiac SR-The proteoliposomes used herein contain both SERCA1a and PLN oriented with their cytoplasmic domains on the external surface of the vesicles at lipid/protein ratios similar to those of cardiac SR membranes (19,29). Although there may be subtle differences in the interaction of PLN with SERCA1a versus SERCA2a (31), the skeletal muscle isoform is well established as a surrogate for structural and functional studies of the complex (for examples, see Refs.…”

“…Mutants were confirmed by DNA sequencing (TAGC Sequencing, University of Alberta) and MALDI-TOF mass spectrometry (Institute for Biomolecular Design, University of Alberta). To phosphorylate PLN, purified PLN was solubilized in detergent and treated with the catalytic subunit of PKA (Sigma-Aldrich) (19).…”

“…Co-reconstitution of SERCA and PLN-SERCA1a was purified from rabbit skeletal muscle SR (20,21) and co-reconstituted with PLN as described (19). The purified proteoliposomes yielded a final molar ratio of 1 SERCA, 4.5 PLN, and 120 lipids.…”