Zhanjia Hou

Isoform Specificity of the Na/K-ATPase Association and Regulation by Phospholemman

Phospholemman (PLM) phosphorylation mediates enhanced Na/K-ATPase (NKA) function during adrenergic stimulation of the heart. Multiple NKA isoforms exist, and their function/regulation may differ. We combined fluorescence resonance energy transfer (FRET) and functional measurements to investigate isoform specificity of the NKA-PLM interaction. FRET was measured as the increase in the donor fluorescence (CFP-NKA-α1 or CFP-NKA-α2) during progressive acceptor (PLM-YFP) photobleach in HEK-293 cells. Both pairs exhibited robust FRET (maximum of 23.6 ± 3.4% for NKA-α1 and 27.5 ± 2.5% for NKA-α2). Donor fluorescence depended linearly on acceptor fluorescence, indicating a 1:1 PLM:NKA stoichiometry for both isoforms. PLM phosphorylation induced by cAMP-dependent protein kinase and protein kinase C activation drastically reduced the FRET with both NKA isoforms. However, submaximal cAMP-dependent protein kinase activation had less effect on PLM-NKA-α2 versus PLM-NKA-α1. Surprisingly, ouabain virtually abolished NKA-PLM FRET but only partially reduced co-immunoprecipitation. PLM-CFP also showed FRET to PLM-YFP, but the relationship during progressive photobleach was highly nonlinear, indicating oligomers involving ≥3 monomers. Using cardiac myocytes from wild-type mice and mice where NKA-α1 is ouabain-sensitive and NKA-α2 is ouabain-resistant, we assessed the effects of PLM phosphorylation on NKA-α1 and NKA-α2 function. Isoproterenol enhanced internal Na+ affinity of both isoforms (K½ decreased from 18.1 ± 2.0 to 11.5 ± 1.9 mm for NKA-α1 and from 16.4 ± 2.5 to 10.4 ± 1.5 mm for NKA-α2) without altering maximum transport rate (Vmax). Protein kinase C activation also decreased K½ for both NKA-α1 and NKA-α2 (to 9.4 ± 1.0 and 9.1 ± 1.1 mm, respectively) but increased Vmax only for NKA-α2 (1.9 ± 0.4 versus 1.2 ± 0.5 mm/min). In conclusion, PLM associates with and modulates both NKA-α1 and NKA-α2 in a comparable but not identical manner.

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 (&tgr;=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 (&tgr;=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.

Phosphomimetic mutations increase phospholamban oligomerization and alter the structure of its regulatory complex.

To investigate the effect of phosphorylation on the interactions of phospholamban (PLB) with itself and its regulatory target, SERCA, we measured FRET from CFP-SERCA or CFP-PLB to YFP-PLB in live AAV-293 cells. Phosphorylation of PLB was mimicked by mutations S16E (PKA site) or S16E/T17E (PKA+CaMKII sites). FRET increased with protein concentration up to a maximum (FRETmax) that was taken to represent the intrinsic FRET of the bound complex. The concentration dependence of FRET yielded dissociation constants (KD) for the PLB-PLB and PLB-SERCA interactions. PLB-PLB FRET data suggest pseudo-phosphorylation of PLB increased oligomerization of PLB but did not alter PLB pentamer quaternary structure. PLB-SERCA FRET experiments showed an apparent decrease in binding of PLB to SERCA and an increase in the apparent PLB-SERCA binding cooperativity. It is likely that these changes are secondary effects of increased oligomerization of PLB; a change in the inherent affinity of monomeric PLB for SERCA was not detected. In addition, PLB-SERCA complex FRETmax was reduced by phosphomimetic mutations, suggesting the conformation of the regulatory complex is significantly altered by PLB phosphorylation.

Lethal Arg9Cys phospholamban mutation hinders Ca2+-ATPase regulation and phosphorylation by protein kinase A

The regulatory interaction of phospholamban (PLN) with Ca2+-ATPase controls the uptake of calcium into the sarcoplasmic reticulum, modulating heart muscle contractility. A missense mutation in PLN cytoplasmic domain (R9C) triggers dilated cardiomyopathy in humans, leading to premature death. Using a combination of biochemical and biophysical techniques both in vitro and in live cells, we show that the R9C mutation increases the stability of the PLN pentameric assembly via disulfide bridge formation, preventing its binding to Ca2+-ATPase as well as phosphorylation by protein kinase A. These effects are enhanced under oxidizing conditions, suggesting that oxidative stress may exacerbate the cardiotoxic effects of the PLNR9C mutant. These results reveal a regulatory role of the PLN pentamer in calcium homeostasis, going beyond the previously hypothesized role of passive storage for active monomers.

Phospholamban Oligomerization, Quaternary Structure, and Sarco(endo)plasmic Reticulum Calcium ATPase Binding Measured by Fluorescence Resonance Energy Transfer in Living Cells

Phospholamban (PLB) oligomerization, quaternary structure, and sarco(endo)plasmic reticulum calcium ATPase (SERCA) binding were quantified by fluorescence resonance energy transfer (FRET) in an intact cellular environment. FRET between cyan fluorescent protein-PLB and yellow fluorescent protein-PLB in AAV-293 cells showed hyperbolic dependence on protein concentration, with a maximum efficiency of 45.1 ± 1.3%. The observed FRET corresponds to a probe separation distance of 58.7 ± 0.5Å, according to a computational model of intrapentameric FRET. This is consistent with models of the PLB pentamer in which cytoplasmic domains fan out from the central bundle of transmembrane helices. An I40A mutation of PLB did not alter pentamer conformation but increased the concentration of half-maximal FRET (KD) by >4-fold. This is consistent with the previous observation that this putatively monomeric mutant still oligomerizes in intact membranes but forms more dynamic pentamers than wild type PLB. PLB association with SERCA, measured by FRET between cyan fluorescent protein-SERCA and yellow fluorescent protein-PLB, was increased by the I40A mutation without any detectable change in probe separation distance. The data indicate that the regulatory complex conformation is not altered by the I40A mutation. A naturally occurring human mutation (L39Stop) greatly reduced PLB oligomerization and SERCA binding and caused mislocalization of PLB to the cytoplasm and nucleus. Overall, the data suggest that the PLB pentamer adopts a “pinwheel” shape in cell membranes, as opposed to a more compact “bellflower” conformation. I40A mutation decreases oligomerization and increases PLB binding to SERCA. Truncation of the transmembrane domain by L39Stop mutation prevents anchoring of the protein in the membrane, greatly reducing PLB binding to itself or its regulatory target, SERCA.

Phospholamban binds with differential affinity to calcium pump conformers

To investigate the mechanism of regulation of sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) by phospholamban (PLB), we expressed Cerulean-SERCA and yellow fluorescent protein (YFP)-PLB in adult rabbit ventricular myocytes using adenovirus vectors. SERCA and PLB were localized in the sarcoplasmic reticulum and were mobile over multiple sarcomeres on a timescale of tens of seconds. We also observed robust fluorescence resonance energy transfer (FRET) from Cerulean-SERCA to YFP-PLB. Electrical pacing of cardiac myocytes elicited cytoplasmic Ca2+ elevations, but these increases in Ca2+ produced only modest changes in SERCA-PLB FRET. The data suggest that the regulatory complex is not disrupted by elevations of cytosolic calcium during cardiac contraction (systole). This conclusion was also supported by parallel experiments in heterologous cells, which showed that FRET was reduced but not abolished by calcium. Thapsigargin also elicited a small decrease in PLB-SERCA binding affinity. We propose that PLB is not displaced from SERCA by high calcium during systole, and relief of functional inhibition does not require dissociation of the regulatory complex. The observed modest reduction in the affinity of the PLB-SERCA complex with Ca2+ or thapsigargin suggests that the binding interface is altered by SERCA conformational changes. The results are consistent with multiple modes of PLB binding or alternative binding sites.

2-Color Calcium Pump Reveals Closure of the Cytoplasmic Headpiece with Calcium Binding

The sarco(endo)plasmic reticulum calcium ATPase (SERCA) undergoes conformational changes while transporting calcium, but the details of the domain motions are still unclear. The objective of the present study was to measure distances between the cytoplasmic domains of SERCA2a in order to reveal the magnitude and direction of conformational changes. Using fluorescence microscopy of live cells, we measured intramolecular fluorescence resonance energy transfer (FRET) from a donor fluorescent protein fused to the SERCA N-terminus to an acceptor fluorescent protein fused to either the N-, P-, or transmembrane domain. The “2-color” SERCA constructs were catalytically active as indicated by ATPase activity in vitro and Ca uptake in live cells. All constructs exhibited dynamic FRET changes in response to the pump ligands calcium and thapsigargin (Tg). These FRET changes were quantified as an index of SERCA conformational changes. Intramolecular FRET decreased with Tg for the two N-domain fusion sites (at residue 509 or 576), while the P- (residue 661) and TM-domain (C-terminus) fusions showed increased FRET with Tg. The magnitude of the Tg-dependent conformational change was not decreased by coexpression of phospholamban (PLB), nor did PLB slow the kinetics of Tg binding. FRET in ionophore-permeabilized cells was lower in EGTA than in saturating calcium for all constructs, indicating a decrease in domain separation distance with the structural transition from E2 (Ca-free) to E1 (Ca-bound). The data suggest closure of the cytoplasmic headpiece with Ca-binding. The present results provide insight into the structural dynamics of the Ca-ATPase. In addition, the 2-color SERCA constructs developed for this study may be useful for evaluating candidate small molecule regulators of Ca uptake activity.

Phosphorylated Phospholamban Stabilizes a Compact Conformation of the Cardiac Calcium-ATPase

The sarcoendoplasmic reticulum calcium ATPase (SERCA) plays a key role in cardiac calcium handling and is considered a high-value target for the treatment of heart failure. SERCA undergoes conformational changes as it harnesses the chemical energy of ATP for active transport. X-ray crystallography has provided insight into SERCA structural substates, but it is not known how well these static snapshots describe in vivo conformational dynamics. The goals of this work were to quantify the direction and magnitude of SERCA motions as the pump performs work in live cardiac myocytes, and to identify structural determinants of SERCA regulation by phospholamban. We measured intramolecular fluorescence resonance energy transfer (FRET) between fluorescent proteins fused to SERCA cytoplasmic domains. We detected four discrete structural substates for SERCA expressed in cardiac muscle cells. The relative populations of these discrete states oscillated with electrical pacing. Low FRET states were most populated in low Ca (diastole), and were indicative of an open, disordered structure for SERCA in the E2 (Ca-free) enzymatic substate. High FRET states increased with Ca (systole), suggesting rigidly closed conformations for the E1 (Ca-bound) enzymatic substates. Notably, a special compact E1 state was observed after treatment with β-adrenergic agonist or with coexpression of phosphomimetic mutants of phospholamban. The data suggest that SERCA calcium binding induces the pump to undergo a transition from an open, dynamic conformation to a closed, ordered structure. Phosphorylated phospholamban stabilizes a unique conformation of SERCA that is characterized by a compact architecture.

Dysferlin Forms a Dimer Mediated by the C2 Domains and the Transmembrane Domain In Vitro and in Living Cells

Dysferlin was previously identified as a key player in muscle membrane repair and its deficiency leads to the development of muscular dystrophy and cardiomyopathy. However, little is known about the oligomerization of this protein in the plasma membrane. Here we report for the first time that dysferlin forms a dimer in vitro and in living adult skeletal muscle fibers isolated from mice. Endogenous dysferlin from rabbit skeletal muscle exists primarily as a ∼460 kDa species in detergent-solubilized muscle homogenate, as shown by sucrose gradient fractionation, gel filtration and cross-linking assays. Fluorescent protein (YFP) labeled human dysferlin forms a dimer in vitro, as demonstrated by fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analyses. Dysferlin also dimerizes in living cells, as probed by fluorescence resonance energy transfer (FRET). Domain mapping FRET experiments showed that dysferlin dimerization is mediated by its transmembrane domain and by multiple C2 domains. However, C2A did not significantly contribute to dimerization; notably, this is the only C2 domain in dysferlin known to engage in a Ca-dependent interaction with cell membranes. Taken together, the data suggest that Ca-insensitive C2 domains mediate high affinity self-association of dysferlin in a parallel homodimer, leaving the Ca-sensitive C2A domain free to interact with membranes.

Relative Affinity of Calcium Pump Isoforms for Phospholamban Quantified by Fluorescence Resonance Energy Transfer

To investigate the regulation of SERCA1a [sarco(endo)plasmic reticulum calcium ATPase] and SERCA2a calcium pump isoforms by phospholamban (PLB), we quantified PLB-SERCA interactions by fluorescence resonance energy transfer (FRET) in live cells. For both SERCA1a and SERCA2a, FRET to PLB increased with increasing protein expression level to a maximum value corresponding to a probe separation distance of 64 A. The data indicate that the respective regulatory complexes assume the same overall quaternary conformation. However, FRET measurements also revealed that PLB has a 50% higher apparent affinity for SERCA1a relative to SERCA2a. The results suggest that despite the structural similarities of the respective regulatory complexes, there is preferential binding of PLB to SERCA1a over SERCA2a. This apparent selectivity may have implications for biochemical studies in which SERCA1a is used as a substitute for SERCA2a. It may also be an important strategic consideration for therapeutic overexpression of SERCA isoforms in cardiac muscle.