Defining the Molecular Components of Calcium Transport Regulation in a Reconstituted Membrane System ² Laxma G. Reddy,* ,‡ Razvan L. Cornea, ‡ Deborah L. Winters, ‡ Edward McKenna, § and David D. Thomas ‡ Department of Biochemistry, Molecular Biology and Biophysics, UniVersity of Minnesota, Minneapolis, Minnesota 55455 and Merck Research Laboratories, West Point, PennsylVania 19486 ReceiVed October 11, 2002; ReVised Manuscript ReceiVed February 19, 2003 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. Phospholamban (PLB) 1 is a small membrane protein that regulates the Ca-ATPase in cardiac sarcoplasmic reticulum (SR) (1-4). In its unphosphorylated form, PLB inhibits the rate of Ca-pumping and ATP hydrolysis by the Ca-ATPase at subsaturating [Ca]. Upon PLB phosphorylation by cAMP- dependent protein kinase A (PKA) or Ca/calmodulin de- pendent protein kinase II (CaM kinase II), this inhibition is relieved (2, 4, 5). Although PLB is not present in fast-twitch muscle (6, 7), PLB has been shown to regulate the fast-twitch Ca-ATPase isoform (SERCA1), as well as the cardiac isoform (SERCA2a), in both reconstitution (8-13) and coexpression (14) experiments. Studies using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and low-angle laser light scattering in SDS solution have shown that wild-type PLB (WT-PLB) is predominantly pentameric, in equilibrium with a minor monomeric fraction (15, 16). Using electron paramagnetic resonance (EPR) spectroscopy, it has been shown that PLB exists in an average oligomeric size of 3.5 in a lipid membrane, and that upon phosphorylation the average oligomeric size increases to 5.3 (17). That study suggested the existence of a dynamic equilibrium between PLB subunits in the lipid bilayer that is regulated by phosphorylation, and that the regulation of PLB’s oligomeric state is critical for its regulation of the Ca-ATPase, with the PLB monomer being the inhibitory species. Alanine-scanning mutagenesis has shown that replacement of single hydro- phobic amino acid residues, Leu or Ile, by Ala, at specific sites in the membrane-spanning region, decreases the stability of PLB pentamers on SDS-PAGE (16), and these effects were confirmed in lipid membranes by EPR (17) and fluorescence (19). Fluorescence has also shown that the Ca- ATPase binds preferentially to the monomeric species of PLB (20). In addition, most mutations that destabilize PLB pentamers have been shown to enhance PLB’s inhibitory potency, supporting the hypothesis that the active inhibitory species of PLB is the monomer, and that increased oligomeric stability of PLB upon phosphorylation contributes to relief of the inhibition (11, 17, 18, 21, 22). However, some transmembrane PLB mutants have inhibi- tory potencies that do not correlate well with their oligomeric states (23, 24), and several mutants outside the transmem- brane domain of PLB (e.g., N27A-PLB and N30A-PLB) have enhanced inhibitory potency despite having pentameric stability comparable to that of WT-PLB (21, 22, 25). On the basis of these results and others (26), it has been proposed that the inhibitory potency of PLB depends on both the self- dissociation constant of the PLB pentamer and the association constant of the PLB monomer with the Ca-ATPase (11, 27). ² This work was supported in part by grants to LGR (NIH 1K02 HL04209, AHA # 0150060N, National Center), DDT (NIH GM27906). * To whom correspondence should be addressed. Tel.: (651)737- 1349. Fax: (651)737-5886. E-mail: lgreddy@mmm.com. ‡ University of Minnesota. § Merck Research Laboratories. 1 Abbreviations: PLB, phospholamban; WT-PLB, wild-type phos- pholamban; N27A-PLB, Asn-27 to Ala mutant of PLB; I40A-PLB, Ile-40 to Ala mutant of PLB; L31A-PLB, Leu-31 to Ala mutant of PLB; C 12E8, octaethylene glycol monododecyl ether; SR, sarcoplasmic reticulum; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; OG, -octyl glucoside; DOPC, dioleoyl phosphatidyl- choline; DOPE, dioleoyl phosphatidylethanolamine; TFE, 2-2-2-tri- fluoroethanol; TFA, trifluoroacetic acid; PKA, cAMP-dependent protein kinase. 4585 Biochemistry 2003, 42, 4585-4592 10.1021/bi026995a CCC: $25.00 © 2003 American Chemical Society Published on Web 03/28/2003