SERCA Regulation Research Articles

Probing the formation of a hetero-dimeric membrane transport complex with dual in vitro and in silico mutagenesis.

The reversible association of transmembrane helices is a fundamental mechanism in how living cells convey information and respond to physiological events. The cardiac calcium transport regulator phospholamban (PLN) is an example of a single-span transmembrane protein that populates a variety of reversible and competing oligomeric states. PLN primarily forms monomers and pentamers in the membrane, where the PLN pentamer is a storage form and the PLN monomer forms a hetero-dimeric inhibitory complex with SERCA. The binding affinity and free-energy of formation of the SERCA-PLN complex in a membrane have not been determined. As is the case for most transmembrane protein interactions, measuring these quantities experimentally is extremely challenging. In this study, we estimated binding affinities by employing in silico alchemical free-energy calculations for all PLN transmembrane alanine substitutions in a membrane bilayer. The binding affinities were calculated separately for the SERCA-PLN complex, a PLN monomer, and a PLN pentamer and compared to in vitro functional measurements of SERCA regulation by the PLN alanine substitutions. Initially, the changes in SERCA inhibition by PLN alanine substitutions were compared to the changes in free energy for the SERCA-PLN complex formed from the PLN monomer. However, the functional data for the PLN alanine substitutions were better explained by the formation of the SERCA-PLN complex directly from the PLN pentamer. This finding points to an inhibitory mechanism favoring conformational selection of SERCA and the interaction of a PLN pentamer with SERCA for 'delivery' of a PLN monomer to the inhibitory site. The implications of these findings suggest that the energetics of helix exchange between homo- and hetero-oligomeric signaling complexes is favored over an intermediate involving a free monomeric helix in the membrane bilayer.

Functional Role of an Unusual Transmembrane Acidic Residue in the Calcium Pump Regulator Myoregulin.

Myoregulin (MLN) is a member of the regulin family, a group of homologous membrane proteins that bind to and regulate the activity of the sarcoplasmic reticulum Ca2+-ATPase (SERCA). MLN, which is expressed in skeletal muscle, contains an acidic residue in its transmembrane domain. The location of this residue, Asp35, is unusual because the relative occurrence of aspartate is very rare (<0.2%) within the transmembrane helix regions. Therefore, we used atomistic simulations and ATPase activity assays of protein co-reconstitutions to probe the functional role of MLN residue Asp35. These structural and functional studies showed Asp35 has no effects on SERCA's affinity for Ca2+ or the structural integrity of MLN in the lipid bilayer. Instead, Asp35 controls SERCA inhibition by populating a bound-like orientation of MLN. We propose Asp35 provides a functional advantage over other members of the regulin family by populating preexisting MLN conformations required for MLN-specific regulation of SERCA. Overall, this study provides new clues about the evolution and functional divergence of the regulin family and offers novel insights into the functional role of acidic residues in transmembrane protein domains.

Complementary in vitro functional studies and in silico alchemical free energy simulations of the sarco-endoplasmic reticulum calcium pump (SERCA) and phospholamban (PLN) complex.

SERCA is ubiquitously expressed in all eukaryotic cells and is responsible for actively transporting calcium ions from the cytoplasm into the SR/ER lumen. PLN is a small transmembrane peptide that regulates SERCA's calcium transport activity in cardiac muscle. Human genetic variants of PLN have been implicated in cardiomyopathies. PLN is a 52 amino acid peptide with an N-terminal cytoplasmic helix (residues 1-17), a short linker region (residues 18-27) and a C-terminal transmembrane helix (residues 28-52). Previous structural studies have identified the binding groove for PLN formed by transmembrane segments M2, M6, and M9 of SERCA (PDB: 4KYT). The structure of the SERCA-PLN complex consists of SERCA in a calcium-free, E1-like state and includes the linker region and transmembrane helix of PLN (residues 20-52). Here, we carry out in vitro functional studies and in silico free energy calculations to examine the effects of alanine scanning mutations in PLN (residues 20-52) on SERCA regulation. From the functional studies, changes in kinetics parameters such as calcium affinity (KCa) and maximal activity (VMax) of SERCA are determined for each of the alanine substitutions. From the in silico alchemical free energy simulations (using Compute Canada supercomputing resources), each of the residues in PLN are alchemically transformed to alanine to determine the change in Gibbs free energy (Δ;Δ;G) relative to the wild-type SERCA-PLN complex. Together, the complementary in vitro and in silico mutational studies provide novel insights into a membrane protein-peptide complex. The PLN variants are quantified in terms of their physiological significance – gain-of-function, loss-of-function, or neutral – and the molecular determinants that control binding specificity.

Abstract P1065: Deletion Of Arginine 14 Increases Phospholamban Oligomerization And Disrupts The Cardiac Force-Frequency Relationship

The Bowditch effect is a physiological phenomenon where accelerated cardiac pacing leads to an increase in the contractile force of the heart. While this has largely been attributed to a frequency-dependent increase in L-type calcium channel influx, evidence suggests intracellular calcium handling proteins must also play an active role. Knock-out of phospholamban (PLB), the inhibitor of the Ca ATPase (SERCA), virtually abolishes the Bowditch effect in mice, suggesting PLB is a major determinant of the cardiac force-frequency relationship. However, no molecular mechanism has explained this frequency-dependent function of PLB. Our lab has previously shown that SERCA loses affinity for PLB when intracellular calcium is elevated. Here, FRET microscopy revealed dynamic decreases in PLB-SERCA binding in response to intracellular calcium elevations. PLB monomers freed from SERCA were rapidly incorporated into PLB pentamers. Interestingly, unbinding of PLB from pentamers was very slow and delayed the recovery of PLB-SERCA complexes by several seconds after calcium elevations. In a computational model of these regulatory binding dynamics, PLB pentamers sequestered inhibitory monomers away from SERCA at fast heart rates. By lowering PLB inhibition of SERCA with increasing cardiac pacing, PLB pentamers serve as a major contributor to the Bowditch effect. Notably, deletion of arginine 14 of PLB causes arrhythmogenic cardiomyopathy and has been thought to destabilize PLB pentamers. Surprisingly, we found R14del-PLB enhanced pentamer stability and slowed the rate of PLB exchange from pentamers to SERCA. We hypothesize that R14del mutation may disrupt frequency-dependent changes in SERCA regulation by PLB during exercise.

Neprilysins regulate muscle contraction and heart function via cleavage of SERCA-inhibitory micropeptides

Muscle contraction depends on strictly controlled Ca2+ transients within myocytes. A major player maintaining these transients is the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase, SERCA. Activity of SERCA is regulated by binding of micropeptides and impaired expression or function of these peptides results in cardiomyopathy. To date, it is not known how homeostasis or turnover of the micropeptides is regulated. Herein, we find that the Drosophila endopeptidase Neprilysin 4 hydrolyzes SERCA-inhibitory Sarcolamban peptides in membranes of the sarcoplasmic reticulum, thereby ensuring proper regulation of SERCA. Cleavage is necessary and sufficient to maintain homeostasis and function of the micropeptides. Analyses on human Neprilysin, sarcolipin, and ventricular cardiomyocytes indicates that the regulatory mechanism is evolutionarily conserved. By identifying a neprilysin as essential regulator of SERCA activity and Ca2+ homeostasis in cardiomyocytes, these data contribute to a more comprehensive understanding of the complex mechanisms that control muscle contraction and heart function in health and disease.

Avian adjustments to cold and non‐shivering thermogenesis: whats, wheres and hows

Avian cold adaptation is hallmarked by innovative strategies of both heat conservation and thermogenesis. While minimizing heat loss can reduce the thermogenic demands of body temperature maintenance, it cannot eliminate the requirement for thermogenesis. Shivering and non-shivering thermogenesis (NST) are the two synergistic mechanisms contributing to endothermy. Birds are of particular interest in studies of NST as they lack brown adipose tissue (BAT), the major organ of NST in mammals. Critical analysis of the existing literature on avian strategies of cold adaptation suggests that skeletal muscle is the principal site of NST. Despite recent progress, isolating the mechanisms involved in avian muscle NST has been difficult as shivering and NST co-exist with its primary locomotory function. Herein, we re-evaluate various proposed molecular bases of avian skeletal muscle NST. Experimental evidence suggests that sarco(endo)plasmic reticulum Ca2+ -ATPase (SERCA) and ryanodine receptor 1 (RyR1) are key in avian muscle NST, through their mediation of futile Ca2+ cycling and thermogenesis. More recent studies have shown that SERCA regulation by sarcolipin (SLN) facilitates muscle NST in mammals; however, its role in birds is unclear. Ca2+ signalling in the muscle seems to be common to contraction, shivering and NST, but elucidating its roles will require more precise measurement of local Ca2+ levels inside avian myofibres. The endocrine control of avian muscle NST is still poorly defined. A better understanding of the mechanistic details of avian muscle NST will provide insights into the roles of these processes in regulatory thermogenesis, which could further inform our understanding of the evolution of endothermy among vertebrates.

Dynamic regulation of SERCA by phospholamban and DWORF

The sarcoplasmic reticulum Ca-ATPase (SERCA) plays a central role in the cardiac cycle and is tightly regulated through interactions with membrane micropeptides. In the heart, phospholamban (PLB) inhibits SERCA, while dwarf open reading frame (DWORF) activates SERCA. Together, these competing regulators adjust Ca transport to meet changing cardiac demands between rest and exercise. Here, we used fluorescence resonance energy transfer (FRET) to measure how these regulatory interactions change as SERCA cycles through different intermediate conformations in its enzymatic cycle.

Homologous cardiac calcium pump regulators phospholamban and sarcolipin adopt distinct oligomeric states in the membrane

Phospholamban (PLN) and Sarcolipin (SLN) are homologous membrane proteins that belong to the family of proteins that regulate the activity of the cardiac calcium pump (sarcoplasmic reticulum Ca2+-ATPase, SERCA). PLN and SLN share highly conserved leucine zipper motifs that control self-association;consequently, it has been proposed that both PLN and SLN assemble into stablepentamers in the membrane.In this study, we used molecular dynamics (MD) simulations and Western blot analysis to investigate the precise molecular architecture of the PLN and SLN oligomers. Analysis showed that the PLN pentamer is the predominant oligomer present in mouse ventricles and ventricle-like human iPSC-derived cardiomyocytes, in agreement with the MD simulations showing stable leucine zipper interactions across all protomer-protomer interfaces and MD replicates. Interestingly, we found that the PLN pentamer populates an asymmetric structure of the transmembrane region, which is likely anintrinsicfeature of the oligomer in a lipid bilayer. The SLN pentamer is not favorably formed across MD replicates and species of origin;instead, SLN from human and mouse atria primarily populate coexisting dimeric and trimeric states. In contrast to previous studies, our findings indicate that the SLN pentamer is not the predominant oligomeric state populated in the membrane. We conclude that despite their structural homology, PLN and SLN adopt distinct oligomeric states in the membrane. We propose that the distinct oligomeric states populated by PLN and SLN may contribute to tissue-specific SERCA regulation via differences in protomer–oligomer exchange, oligomer–SERCA dynamics, and noise filtering during β-adrenergic stimulation in the heart.

Structural basis for allosteric control of the SERCA-Phospholamban membrane complex by Ca2+ and phosphorylation.

Phospholamban (PLN) is a mini-membrane protein that directly controls the cardiac Ca2+-transport response to β-adrenergic stimulation, thus modulating cardiac output during the fight-or-flight response. In the sarcoplasmic reticulum membrane, PLN binds to the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), keeping this enzyme's function within a narrow physiological window. PLN phosphorylation by cAMP-dependent protein kinase A or increase in Ca2+ concentration reverses the inhibitory effects through an unknown mechanism. Using oriented-sample solid-state NMR spectroscopy and replica-averaged NMR-restrained structural refinement, we reveal that phosphorylation of PLN's cytoplasmic regulatory domain signals the disruption of several inhibitory contacts at the transmembrane binding interface of the SERCA-PLN complex that are propagated to the enzyme's active site, augmenting Ca2+ transport. Our findings address long-standing questions about SERCA regulation, epitomizing a signal transduction mechanism operated by posttranslationally modified bitopic membrane proteins.

Conserved Luminal C-Terminal Domain Dynamically Controls Interdomain Communication in Sarcolipin.

Sarcolipin (SLN) mediates Ca2+ transport and metabolism in muscle by regulating the activity of the Ca2+ pump SERCA. SLN has a conserved luminal C-terminal domain that contributes to its functional divergence among homologous SERCA regulators, but the precise mechanistic role of this domain remains poorly understood. We used all-atom molecular dynamics (MD) simulations of SLN totaling 77.5 μs to show that the N- (NT) and C-terminal (CT) domains function in concert. Analysis of the MD simulations showed that serial deletions of the SLN C-terminus do not affect the stability of the peptide nor induce dissociation of SLN from the membrane but promote a gradual decrease in both the tilt angle of the transmembrane helix and the local thickness of the lipid bilayer. Mutual information analysis showed that the NT and CT domains communicate with each other in SLN and that interdomain communication is partially or completely abolished upon deletion of the conserved segment Tyr29-Tyr31 as well as by serial deletions beyond this domain. Phosphorylation of SLN at residue Thr5 also induces changes in the communication between the CT and NT domains, which thus provides additional evidence for interdomain communication within SLN. We found that interdomain communication is independent of the force field used and lipid composition, which thus demonstrates that communication between the NT and CT domains is an intrinsic functional feature of SLN. We propose the novel hypothesis that the conserved C-terminus is an essential element required for dynamic control of SLN regulatory function.

A hallmark of phospholamban functional divergence is located in the N-terminal phosphorylation domain

Sarcoplasmic reticulum Ca2+ pump (SERCA) is a critical component of the Ca2+ transport machinery in myocytes. There is clear evidence for regulation of SERCA activity by PLB, whose activity is modulated by phosphorylation of its N-terminal domain (residues 1–25), but there is less clear evidence for the role of this domain in PLB’s functional divergence. It is widely accepted that only sarcolipin (SLN), a protein that shares substantial homology with PLB, uncouples SERCA Ca2+ transport from ATP hydrolysis by inducing a structural change of its energy-transduction domain; yet, experimental evidence shows that the transmembrane domain of PLB (residues 26–52, PLB26–52) partially uncouples SERCA in vitro. These apparently conflicting mechanisms suggest that PLB’s uncoupling activity is encoded in its transmembrane domain, and that it is controlled by the N-terminal phosphorylation domain. To test this hypothesis, we performed molecular dynamics simulations (MDS) of the binary complex between PLB26–52 and SERCA. Comparison between PLB26–52 and wild-type PLB (PLBWT) showed no significant changes in the stability and orientation of the transmembrane helix, indicating that PLB26–52 forms a native-like complex with SERCA. MDS showed that PLB26–52 produces key intermolecular contacts and structural changes required for inhibition, in agreement with studies showing that PLB26–52 inhibits SERCA. However, deletion of the N-terminal phosphorylation domain facilitates an order-to-disorder shift in the energy-transduction domain associated with uncoupling of SERCA, albeit weaker than that induced by SLN. This mechanistic evidence reveals that the N-terminal phosphorylation domain of PLB is a primary contributor to the functional divergence among homologous SERCA regulators.

Structural Basis of Sarco/Endoplasmic Reticulum Ca2+-ATPase 2b Regulation via Transmembrane Helix Interplay

Sarco/endoplasmic reticulum (ER) Ca2+-ATPase 2b (SERCA2b) is a ubiquitously expressed membrane protein that facilitates Ca2+ uptake from the cytosol to the ER. SERCA2b includes a characteristic 11th transmembrane helix (TM11) followed by a luminal tail, but the structural basis of SERCA regulation by these C-terminal segments remains unclear. Here, we determined the crystal structures of SERCA2b and its C-terminal splicing variant SERCA2a, both in the E1-2Ca2+-adenylyl methylenediphosphonate (AMPPCP) state. Despite discrepancies with the previously reported structural model of SERCA2b, TM11 was found to be located adjacent to TM10 and to interact weakly with a part of the L8/9 loop and theN-terminal end of TM10, thereby inhibiting the SERCA2b catalytic cycle. Accordingly, mutational disruption of the interactions between TM11 and its neighboring residues caused SERCA2b to display SERCA2a-like ATPase activity. We propose that TM11 serves as a key modulator of SERCA2b activity by fine-tuning the intramolecular interactions with other transmembrane regions.

The Phospholamban Pentamer Functionally Interacts with the Sarcoplasmic Reticulum Calcium Pump SERCA

The interaction of phospholamban with the sarcoplasmic reticulum calcium pump (SERCA) is a major regulatory axis in cardiac muscle contractility. The prevailing model involves reversible inhibition of SERCA by monomeric phospholamban and storage of phospholamban as an inactive pentamer. However, this paradigm has been challenged by studies demonstrating that phospholamban remains associated with SERCA and that the phospholamban pentamer is required for the regulation of cardiac contractility. We have previously used two-dimensional crystallization and electron microscopy to study the interaction between SERCA and phospholamban. To further understand this interaction, we compared small helical crystals and large two-dimensional crystals of SERCA in the absence and presence of phospholamban. In both crystal forms, SERCA molecules are organized into identical anti-parallel dimer ribbons. The dimer ribbons pack together with distinct crystal contacts in the helical versus large two-dimensional crystals, which allow phospholamban differential access to potential sites of interaction with SERCA. Nonetheless, we show that a phospholamban oligomer interacts with SERCA in a similar manner in both crystal forms. In the two-dimensional crystals, a phospholamban pentamer interacts with transmembrane segments M3 of SERCA and participates in a crystal contact that bridges neighboring SERCA dimer ribbons. In the helical crystals, an oligomeric form of phospholamban also interacts with M3 of SERCA, though the phospholamban oligomer straddles a SERCA-SERCA crystal contact. We conclude that the pentameric form of phospholamban interacts with M3 of SERCA, and that it plays distinct structural and functional roles in SERCA regulation. The interaction of the pentamer places the cytoplasmic domains of phospholamban at the membrane surface proximal to the calcium entry funnel of SERCA. This interaction may cause localized perturbation of the membrane bilayer as a mechanism for altering the turnover rate of SERCA.

Phospholamban deficiency does not alter skeletal muscle SERCA pumping efficiency or predispose mice to diet-induced obesity.

The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pump is a major contributor to skeletal muscle Ca2+ homeostasis and metabolic rate. SERCA activity can become adaptively uncoupled by its regulator sarcolipin (SLN) to increase the energy demand of Ca2+ pumping, preventing excessive obesity and glucose intolerance in mice. Several other SERCA regulators bear structural and functional resemblance to SLN, including phospholamban (PLN). Here, we sought to examine whether endogenous levels of skeletal muscle PLN control SERCA Ca2+ pumping efficiency and whole body metabolism. Using PLN-null mice ( Pln-/-), we found that soleus (SOL) muscle's SERCA pumping efficiency (measured as an apparent coupling ratio: Ca2+ uptake/ATP hydrolysis) was unaffected by PLN. Expression of Ca2+-handling proteins within the SOL, including SLN, were comparable between Pln-/- and wild-type (WT) littermates, as were fiber-type characteristics. Not surprisingly then, Pln-/- mice developed a similar degree of diet-induced obesity and glucose intolerance as WT controls when given a "Western" high-fat diet. Lack of an excessively obesogenic phenotype of Pln-/- could not be explained by compensation from skeletal muscle SLN or brown adipose tissue uncoupling protein-1 content. In agreement with several other reports, our study lends support to the notion that PLN serves a functionally distinct role from that of SLN in skeletal muscle physiology.

Effects of the Arg9Cys and Arg25Cys mutations on phospholamban's conformational equilibrium in membrane bilayers

Approximately, 70% of the Ca2+ ion transport into the sarcoplasmic reticulum is catalyzed by the sarcoplasmic reticulum Ca2+-ATPase (SERCA), whose activity is endogenously regulated by phospholamban (PLN). PLN comprises a TM inhibitory region and a cytoplasmic regulatory region that harbors a consensus sequence for cAMP-dependent protein kinase (PKA). The inhibitory region binds the ATPase, reducing its apparent Ca2+ binding affinity. β-adrenergic stimulation activates PKA, which phosphorylates PLN at Ser 16, reversing its inhibitory function. Mutations and post-translational modifications of PLN may lead to dilated cardiomyopathy (DCM) and heart failure. PLN's cytoplasmic region interconverts between a membrane-associated T state and a membrane-detached R state. The importance of these structural transitions on SERCA regulation is emerging, but the effects of natural occurring mutations and their relevance to the progression of heart disease are unclear. Here we use solid-state NMR spectroscopy to investigate the structural dynamics of two lethal PLN mutations, R9C and R25C, which lead to DCM. We found that the R25C mutant enhances the dynamics of PLN and shifts the conformational equilibrium toward the R state confirmation, whereas the R9C mutant drives the amphipathic cytoplasmic domain toward the membrane-associate state, enriching the T state population. The changes in membrane interactions caused by these mutations may explain the aberrant regulation of SERCA.

The SarcoEndoplasmic Reticulum Calcium ATPase.

The calcium pump (a.k.a. Ca2+-ATPase or SERCA) is a membrane transport protein ubiquitously found in the endoplasmic reticulum (ER) of all eukaryotic cells. As a calcium transporter, SERCA maintains the low cytosolic calcium level that enables a vast array of signaling pathways and physiological processes (e.g. synaptic transmission, muscle contraction, fertilization). In muscle cells, SERCA promotes relaxation by pumping calcium ions from the cytosol into the lumen of the sarcoplasmic reticulum (SR), the main storage compartment for intracellular calcium. X-ray crystallographic studies have provided an extensive understanding of the intermediate states that SERCA populates as it progresses through the calcium transport cycle. Historically, SERCA is also known to be regulated by small transmembrane peptides, phospholamban (PLN) and sarcolipin (SLN). PLN is expressed in cardiac muscle, whereas SLN predominates in skeletal and atrial muscle. These two regulatory subunits play critical roles in cardiac contractility. While our understanding of these regulatory mechanisms are still developing, SERCA and PLN are one of the best understood examples of peptide-transporter regulatory interactions. Nonetheless, SERCA appeared to have only two regulatory subunits, while the related sodium pump (a.k.a. Na+, K+-ATPase) has at least nine small transmembrane peptides that provide tissue specific regulation. The last few years have seen a renaissance in our understanding of SERCA regulatory subunits. First, structures of the SERCA-SLN and SERCA-PLN complexes revealed molecular details of their interactions. Second, an array of micropeptides concealed within long non-coding RNAs have been identified as new SERCA regulators. This chapter will describe our current understanding of SERCA structure, function, and regulation.

NMR Resolves Phospholamban's Allosteric Regulation of the Sarcoplasmic Reticulum Ca2+-ATPase

Disruption of cardiac calcium cycling due to dysregulation of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) by the small transmembrane protein, phospholamban (PLN) leads to severe phenotypes including arrhythmia and dilated cardiomyopathy (DCM). In order to understand how inherited mutations in PLN lead to these severe phenotypes it is essential to map PLN's binding interaction with SERCA and how this binding interaction is modulated by post-translational modifications in PLN.Here we have applied both magic angle spinning (MAS) and oriented solid-state NMR techniques to characterize the structure, dynamics, and topology of monomeric constructs of PLN in complex with SERCA to better understand how post-translational modifications and mutations in PLN alter SERCA's activity. By combining chemical shift perturbations, dipolar couplings, and other structural restraints with molecular dynamics simulations, we have built a model of the PLN-SERCA complex that outlines a mechanism for PLN's allosteric regulation of SERCA via its cytoplasmic domain.

Combining Solution and Solid State NMR Techniques in the Analysis of Arg to Cys Mutations in Phospholamban Associated with Dilated Cardiomyopathy

Dilated cardiomyopathy (DCM) is characterized by the enlargement and weakening of the left ventricle. This dilation of the ventricle prevents the heart from contracting normally and can spread to the right ventricle and atria as the disease progresses. Inherited forms of DCM have been identified in families that carry missense, deletion, or nonsense mutations in the regulatory protein, phospholamban (PLN). Two of the identified missense mutations, R9C and R25C, result in cysteine substitutions for arginine residues in PLN's cytoplasmic domain. In vitro studies with these mutants indicate that they have an altered ability to regulate SERCA, however it is unclear how these mutations lead to dysregulation of SERCA.Here we have applied both solution and solid-state NMR techniques to characterize the structure, dynamics and topology of the monomeric constructs for the R9C and R25C mutants to better understand how these mutations affect PLN's regulation of SERCA in the development of DCM. These studies were pursued under both reducing and oxidizing conditions to better reflect the physiological conditions of DCM progression and for comparison to coupled enzyme activity assays used to quantify the effect of these mutants on SERCA activity. We have utilized chemical shift perturbations as sensitive probes toward the local environment of the cytoplasmic and juxta-membrane domains of PLN which harbor the aforementioned mutations. Dynamics in lipid bilayers have been probed through cross-polarization or J-coupling enabled experiments which are exclusively sensitive toward either the rigid or mobile regions of the protein. Heteronuclear NMR was employed to probe the fast timescale dynamics of these mutants. Finally, separated local field experiments in magnetically aligned bicelles were employed to probe the topology.

Correlating Structure and Function in Human Genetic Variants of Phospholamban and Sarcolipin

Calcium is an essential signal carrier that enables cellular processes such as muscle contraction, secretion, and metabolism. Consequently, calcium dysregulation is associated with many human diseases, including heart failure, cancer, diabetes, and many others. Considering heart failure alone, 26 million people currently suffer worldwide. The principal site for cellular calcium homeostasis is the endo- and sarcoplasmic reticulum, where resident calcium pumps (SERCA) actively maintain cytosolic calcium. SERCA is so important for cellular function that it is controlled by a number of tissue-specific master regulators – phospholamban (PLN), sarcolipin (SLN) and myoregulin (MLN). These SERCA regulators control cardiac contractility, thermogenesis, and skeletal muscle performance. Herein we present mutagenesis and structure-function studies of PLN and SLN, with an emphasis on clinically relevant mutations. As genome sequencing projects rapidly expand the spectrum of human genetic variants in PLN and SLN, this information provides a basis for targeted structure-function analyses. Focusing on missense variants and chimeras of these peptides, we use membrane reconstitution, functional assays and biophysical approaches – CD spectroscopy of PLN and SLN variants and electron cryo-microscopy (cryoEM) of two-dimensional crystals of SERCA-PLN and SERCA-SLN. Some of the mutants investigated include Ala15-Thr, Pro21-Thr, and Leu51-Pro. The Ala15-Thr mutant has comparable inhibitory activity to wild-type PLN, but the interaction with protein kinase A is altered. The Pro21-Thr mutant has comparable inhibitory activity to wild-type PLN, but it has a more helical conformation reminiscent of early models for the PLN pentamer. Finally, the Leu51-Pro mutant is a less helical form of PLN with complete loss of inhibitory function. Since Pro21-Thr and Leu51-Pro alter PLN structure, they are ideal candidates for two-dimensional crystallization with SERCA and cryoEM. These studies are underway, and our current progress on PLN, SLN and MLN will be presented.

Aldosterone Impairs Vascular Smooth Muscle Function: From Clinical to Bench Research.

The effect of aldosterone on vascular smooth muscle cell function is still unclear. One method to measure vascular smooth muscle cell function is endothelial-independent vascular dilation, for which the key factor is sarcoplasmic reticulum calcium adenosine triphosphatase (SERCA). Our objective was to investigate the effect of aldosterone on vascular smooth muscle cell function and SERCA regulation. We prospectively analyzed 35 patients with primary aldosteronism (PA; 32 patients with aldosterone-producing adenoma and three patients with idiopathic hyperaldosteronism) and 30 patients with essential hypertension (EH) who were enrolled as the control group. Flow and nitrate-mediated dilation were performed in both groups and 1 year after adrenalectomy in the patients with aldosterone-producing adenoma. In addition, we investigated the effect of aldosterone on SERCA regulation in human aortic smooth muscle cells. This study took place in an academic clinical research center. Participants included 35 patients with PA and 30 patients with EH. Adrenalectomy was undertaken in patients with aldosterone-producing adenoma. The PA patients had significantly lower flow-mediated dilation (FMD) and nitrate-mediated dilation (NMD) values than the patients with EH (FMD: 13 ± 6 vs 16 ± 4; NMD: 16 ± 6 vs 19 ± 5; both P < .05). FMD/NMD were significantly correlated with log 24 hour-urine aldosterone (FMD: r = -0.287, P = .048; NMD: r = -0.402, P = .005) but not blood pressure. The impaired FMD and NMD values were significantly restored 1 year after adrenalectomy (FMD: 11 ± 4 to 19 ± 7; NMD: 15 ± 6 to 21 ± 6; both P < .01). Under confocal microscopy, aldosterone was shown to suppress the expression of SERCA2a of human aortic smooth muscle cells. Aldosterone significantly suppressed the expression of SERCA2a from 10(-8) M in mRNA and protein levels. This suppression was through down-regulation of mineralocorticoid receptor dependent mitochondrial transcription factors A and B2. Aldosterone impairs vascular smooth muscle cell function and suppresses SERCA 2a expression.