Oligomeric interactions of sarcolipin and the Ca-ATPase.

We have detected directly the interactions of sarcolipin (SLN) and the sarcoplasmic reticulum Ca-ATPase (SERCA) by measuring fluorescence resonance energy transfer (FRET) between fusion proteins labeled with cyan fluorescent protein (donor) and yellow fluorescent protein (acceptor). SLN is a membrane protein that helps control contractility by regulating SERCA activity in fast-twitch and atrial muscle. Here we used FRET microscopy and spectroscopy with baculovirus expression in insect cells to provide direct evidence for: 1) oligomerization of SLN and 2) regulatory complex formation between SLN and the fast-twitch muscle Ca-ATPase (SERCA1a isoform). FRET experiments demonstrated that SLN monomers self-associate into dimers and higher order oligomers in the absence of SERCA, and that SLN monomers also bind to SERCA monomers in a 1:1 binary complex when the two proteins are coexpressed. FRET experiments further demonstrated that the binding affinity of SLN for itself is similar to that for SERCA. Mutating SLN residue isoleucine-17 to alanine (I17A) decreased the binding affinity of SLN self-association and converted higher order oligomers into monomers and dimers. The I17A mutation also decreased SLN binding affinity for SERCA but maintained 1:1 stoichiometry in the regulatory complex. Thus, isoleucine-17 plays dual roles in determining the distribution of SLN homo-oligomers and stabilizing the formation of SERCA-SLN heterodimers. FRET results for SLN self-association were supported by the effects of SLN expression in bacterial cells. We propose that SLN exists as multiple molecular species in muscle, including SERCA-free (monomer, dimer, oligomer) and SERCA-bound (heterodimer), with transmembrane zipper residues of SLN serving to stabilize oligomeric interactions.

Interdomain fluorescence resonance energy transfer in SERCA probed by cyan-fluorescent protein fused to the actuator domain.

We have used a biosynthetically incorporated fluorescent probe to monitor domain movements involved in ion transport by the sarcoendoplasmic reticulum Ca-ATPase (SERCA) from rabbit fast-twitch skeletal muscle. X-ray crystal structures suggest that the nucleotide-binding (N) and actuator (A) domains of SERCA move apart by several nanometers upon Ca binding. To test this hypothesis, cDNA constructs were created to fuse cyan-fluorescent protein (CFP) to the N terminus of SERCA (A domain). This CFP-SERCA fluorescent fusion protein retained activity when expressed in Sf21 insect cells using the baculovirus system. Fluorescence resonance energy transfer (FRET) was used to monitor the A-N interdomain distance for CFP-SERCA selectively labeled with fluorescein isothiocyanate (FITC) at Lys 515 in the N domain. At low [Ca (2+)] (E2 biochemical state), the measured FRET efficiency between CFP (donor in A domain) and FITC (acceptor in N domain) was 0.34 +/- 0.03, indicating a mean distance of 61.6 +/- 2.0 A between probes on the two domains. An increase of [Ca (2+)] to 0.1 mM (E1-Ca biochemical state) decreased the FRET efficiency by 0.06 +/- 0.03, indicating an increase in the mean distance by 3.0 +/- 1.2 A. Quantitative molecular modeling of dual-labeled SERCA, including an accurate calculation of the orientation factor, shows that the FRET data observed in the absence of Ca is consistent with the E2 crystal structure, but the increase in distance (decrease in FRET) induced by Ca is much less than predicted by the E1 crystal structure. We conclude that the E1 crystal structure does not reflect the predominant structure of SERCA under physiological conditions in a functional membrane bilayer.

Förster transfer recovery reveals that phospholamban exchanges slowly from pentamers but rapidly from the SERCA regulatory complex.

Phospholamban (PLB) or the sarcoplasmic reticulum Ca2+-ATPase (SERCA) were fused to cyan fluorescent protein (CFP) and coexpressed with PLB fused to yellow fluorescent protein (YFP). The expressed fluorescently tagged proteins were imaged using epifluorescence and total internal reflection fluorescence microscopy. YFP fluorescence was selectively bleached by a focused laser beam. CFP fluorescence at the targeted site increased after YFP photobleaching, indicating fluorescence resonance energy transfer between CFP-SERCA/CFP-PLB and YFP-PLB. The increased donor fluorescence relaxed back toward baseline as a result of donor diffusion and exchange of bleached YFP-PLB for unbleached YFP-PLB, which restored fluorescence resonance energy transfer. Requenching of CFP donors, termed Förster transfer recovery (FTR), was quantified as an index of the rate of PLB subunit exchange from the PLB:SERCA and PLB:PLB membrane complexes. PLB subunit exchange from the PLB:SERCA regulatory complex was rapid, showing diffusion-limited FTR (tau=1.4 second). Conversely, PLB:PLB oligomeric complexes were found to be stable on a much longer time scale. Despite free lateral diffusion in the membrane, they showed no FTR over 80 seconds. Mutation of PLB position 40 from isoleucine to alanine (I40A-PLB) did not abolish PLB:PLB energy transfer, but destabilization of the PLB:PLB complex was apparent from an increased FTR rate (tau=8.4 seconds). Oligomers of I40A-PLB were stabilized by oxidative crosslinking of transmembrane cysteines with diamide. We conclude that PLB exchanges rapidly from its regulatory complex with the SERCA pump, whereas subunit exchange from the PLB oligomeric complex is slow and does not occur on the time scale of the cardiac cycle.

Defining the molecular components of calcium transport regulation in a reconstituted membrane system.

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.