Peter Vangheluwe

Sarcolipin and phospholamban mRNA and protein expression in cardiac and skeletal muscle of different species

The widely held view that SLN (sarcolipin) would be the natural inhibitor of SERCA1 (sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase 1), and PLB (phospholamban) its counterpart for SERCA2 inhibition is oversimplified and partially wrong. The expression of SLN and PLB mRNA and protein relative to SERCA1 or SERCA2 was assessed in ventricle, atrium, soleus and EDL (extensor digitorum longus) of mouse, rat, rabbit and pig. SLN protein levels were quantified by means of Western blotting using what appears to be the first successfully generated antibody directed against SLN. Our data confirm the co-expression of PLB and SERCA2a in cardiac muscle and the very low levels (in pig and rabbit) or the absence (in rat and mouse) of PLB protein in the slow skeletal muscle. In larger animals, the SLN mRNA and protein expression in the soleus and EDL correlates with SERCA1a expression, but, in rodents, SLN mRNA and protein show the highest abundance in the atria, which are devoid of SERCA1. In the rodent atria, SLN could therefore potentially interact with PLB and SERCA2a. No SLN was found in the ventricles of the different species studied, and there was no compensatory SLN up-regulation for the loss of PLB in PLB(-/-) mouse. In addition, we found that SLN expression was down-regulated at the mRNA and protein level in the atria of hypertrophic hearts of SERCA2(b/b) mice. These data suggest that superinhibition of SERCA by PLB-SLN complexes could occur in the atria of the smaller rodents, but not in those of larger animals.

The Ca2+ Pumps of the Endoplasmic Reticulum and Golgi Apparatus

The various splice variants of the three SERCA- and the two SPCA-pump genes in higher vertebrates encode P-type ATPases of the P(2A) group found respectively in the membranes of the endoplasmic reticulum and the secretory pathway. Of these, SERCA2b and SPCA1a represent the housekeeping isoforms. The SERCA2b form is characterized by a luminal carboxy terminus imposing a higher affinity for cytosolic Ca(2+) compared to the other SERCAs. This is mediated by intramembrane and luminal interactions of this extension with the pump. Other known affinity modulators like phospholamban and sarcolipin decrease the affinity for Ca(2+). The number of proteins reported to interact with SERCA is rapidly growing. Here, we limit the discussion to those for which the interaction site with the ATPase is specified: HAX-1, calumenin, histidine-rich Ca(2+)-binding protein, and indirectly calreticulin, calnexin, and ERp57. The role of the phylogenetically older and structurally simpler SPCAs as transporters of Ca(2+), but also of Mn(2+), is also addressed.

Sarcoplasmic reticulum calcium uptake and speed of relaxation are depressed in nebulin-free skeletal muscle

Previous work suggested that altered Ca2+ homeostasis might contribute to dysfunction of nebulin‐free muscle, as gene expression analysis revealed that the sarco(endo)plasmic reticulum Ca2+‐ATPase (SERCA)‐inhibitor sarcolipin (SLN) is up‐regulated > 70‐fold in nebulin knockout mice, and here we tested this proposal. We investigated SLN protein expression in nebulin‐free and wild‐type skeletal muscle, as well as expression of other Ca2+‐handling proteins. Ca2+ uptake capacity was determined in isolated sarcoplasmic reticulum vesicles and in intact myofibers by measuring Ca2+ transients. Muscle contractile performance was determined in skinned muscle activated with exogenous Ca2+, as well as in electrically stimulated intact muscle. We found profound up‐regulation of SLN protein in nebulin‐free skeletal muscle, whereas expression of other Ca2+‐handling proteins was not (calsequestrin and phospholamban) or was minimally (SERCA) affected. Speed of Ca2+ uptake was >3‐fold decreased in sarcoplasmic reticulum vesicles isolated from nebulin‐free muscle as well as in nebulin‐free intact myofibers. Ca2+‐activated stress in skinned muscle and stress produced by intact nebulin‐free muscle were reduced to a similar extent compared with wild type. Half‐relaxation time was significantly longer in nebulin‐free compared with wild‐type muscle. Thus, the present study demonstrates for the first time that nebulin might also be involved in physiological Ca2+ handling of the SR‐myofibrillar system.—Ottenheijm, C. A. C., Fong, C., Vangheluwe, P., Wuytack, F., Babu, G. J., Periasamy, M., Witt, C. C., Labeit, S., Granzier, H. Sarcoplasmic reticulum calcium uptake and speed of relaxation are depressed in nebulin‐free skeletal muscle. FASEB J. 22, 2912–2919 (2008)

Intracellular Ca2+- and Mn2+-Transport ATPases

3.4.3. Artemisinin L 4. SPCAs L 4.1. Genes Encoding SPCAs L 4.2. Structure of SPCAs M 4.2.1. Mn2+ and Ca2+ Binding M 4.2.2. Countertransport M 4.3. Expression of SPCAs N 4.3.1. SPCA1 N 4.3.2. SPCA2 N 4.4. Kinetic Properties of SPCAs N 4.5. SPCA Inhibitors O 4.6. Function of SPCAs O 4.6.1. pmr1 Mutants in Yeast O 4.6.2. RNA Interference O 4.6.3. SPCA1 Mouse Models P 5. Other Ca2+and Mn2+-Transporting P-type ATPases P

Structural basis for the high Ca2+ affinity of the ubiquitous SERCA2b Ca2+ pump

Sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) Ca2+ transporters pump cytosolic Ca2+ into the endoplasmic reticulum, maintaining a Ca2+ gradient that controls vital cell functions ranging from proliferation to death. To meet the physiological demand of the cell, SERCA activity is regulated by adjusting the affinity for Ca2+ ions. Of all SERCA isoforms, the housekeeping SERCA2b isoform displays the highest Ca2+ affinity because of a unique C-terminal extension (2b-tail). Here, an extensive structure–function analysis of SERCA2b mutants and SERCA1a2b chimera revealed how the 2b-tail controls Ca2+ affinity. Its transmembrane (TM) segment (TM11) and luminal extension functionally cooperate and interact with TM7/TM10 and luminal loops of SERCA2b, respectively. This stabilizes the Ca2+-bound E1 conformation and alters Ca2+-transport kinetics, which provides the rationale for the higher apparent Ca2+ affinity. Based on our NMR structure of TM11 and guided by mutagenesis results, a structural model was developed for SERCA2b that supports the proposed 2b-tail mechanism and is reminiscent of the interaction between the α- and β-subunits of Na+,K+-ATPase. The 2b-tail interaction site may represent a novel target to increase the Ca2+ affinity of malfunctioning SERCA2a in the failing heart to improve contractility.

Ca2+ transport ATPase isoforms SERCA2a and SERCA2b are targeted to the same sites in the murine heart.

Adult SERCA2(b/b) mice expressing the non-muscle Ca2+ transport ATPase isoform SERCA2b in the heart instead of the normally predominant sarcomeric SERCA2a isoform, develop mild concentric ventricular hypertrophy with impaired cardiac contractility and relaxation [Circ. Res. 89 (2001) 838]. Results from a separate study on transgenic mice overexpressing SERCA2b in the normal SERCA2a context were interpreted to show that SERCA2b and SERCA2a are differentially targeted within the cardiac sarcoplasmic reticulum (SR) [J. Biol. Chem. 275 (2000) 24722]. Since a different subcellular distribution of SERCA2b could underlie alterations in Ca2+ handling observed in SERCA2(b/b), we wanted to compare SERCA2b distribution in SERCA2(b/b) with that of SERCA2a in wild-type (WT). Using confocal microscopy on immunostained fixed myocytes and BODIPY-thapsigargin-stained living cells, we found that in SERCA2(b/b) mice SERCA2b is correctly targeted to cardiac SR and is present in the same SR regions as SERCA2a and SERCA2b in WT. We conclude that there is no differential targeting of SERCA2a and SERCA2b since both are found in the longitudinal SR and in the SR proximal to the Z-bands. Therefore, alterations in Ca2+ handling and the development of hypertrophy in adult SERCA2(b/b) mice do not result from different SERCA2b targeting.

Sarco(endo)plasmic Reticulum Calcium ATPase (SERCA) Inhibition by Sarcolipin Is Encoded in Its Luminal Tail

Background: Sarcolipin is a regulator of SERCA in skeletal and atrial muscle with inhibitory properties thought to be similar to phospholamban. Results: Residues critical for SERCA inhibition reside in the luminal extension of sarcolipin. Conclusion: The luminal extension of sarcolipin is a distinct and transferrable domain that encodes most of its inhibitory properties. Significance: Sarcolipin and phospholamban use different inhibitory mechanisms to regulate SERCA. The sarco(endo)plasmic reticulum calcium ATPase (SERCA) is regulated in a tissue-dependent manner via interaction with the short integral membrane proteins phospholamban (PLN) and sarcolipin (SLN). Although defects in SERCA activity are known to cause heart failure, the regulatory mechanisms imposed by PLN and SLN could have clinical implications for both heart and skeletal muscle diseases. PLN and SLN have significant sequence homology in their transmembrane regions, suggesting a similar mode of binding to SERCA. However, unlike PLN, SLN has a conserved C-terminal luminal tail composed of five amino acids (27RSYQY), which may contribute to a distinct SERCA regulatory mechanism. We have functionally characterized alanine mutants of the C-terminal tail of SLN using co-reconstituted proteoliposomes of SERCA and SLN. We found that Arg27 and Tyr31 are essential for SLN function. We also tested the effect of a truncated variant of SLN (Arg27stop) and extended chimeras of PLN with the five luminal residues of SLN added to its C terminus. The Arg27stop form of SLN resulted in loss of function, whereas the PLN chimeras resulted in superinhibition with characteristics of both PLN and SLN. Based on our results, we propose that the C-terminal tail of SLN is a distinct, essential domain in the regulation of SERCA and that the functional properties of the SLN tail can be transferred to PLN.

Cellular function and pathological role of ATP13A2 and related P-type transport ATPases in Parkinson's disease and other neurological disorders.

Mutations in ATP13A2 lead to Kufor-Rakeb syndrome, a parkinsonism with dementia. ATP13A2 belongs to the P-type transport ATPases, a large family of primary active transporters that exert vital cellular functions. However, the cellular function and transported substrate of ATP13A2 remain unknown. To discuss the role of ATP13A2 in neurodegeneration, we first provide a short description of the architecture and transport mechanism of P-type transport ATPases. Then, we briefly highlight key P-type ATPases involved in neuronal disorders such as the copper transporters ATP7A (Menkes disease), ATP7B (Wilson disease), the Na+/K+-ATPases ATP1A2 (familial hemiplegic migraine) and ATP1A3 (rapid-onset dystonia parkinsonism). Finally, we review the recent literature of ATP13A2 and discuss ATP13A2s putative cellular function in the light of what is known concerning the functions of other, better-studied P-type ATPases. We critically review the available data concerning the role of ATP13A2 in heavy metal transport and propose a possible alternative hypothesis that ATP13A2 might be a flippase. As a flippase, ATP13A2 may transport an organic molecule, such as a lipid or a peptide, from one membrane leaflet to the other. A flippase might control local lipid dynamics during vesicle formation and membrane fusion events.

Factors controlling the activity of the SERCA2a pump in the normal and failing heart

Heart failure is the leading cause of death in western countries and is often associated with impaired Ca2+ handling in the cardiomyocyte. In fact, cardiomyocyte relaxation and contraction are tightly controlled by the activity of the cardiac sarco(endo)plasmic reticulum (ER/SR) Ca2+ pump SERCA2a, pumping Ca2+ from the cytosol into the lumen of the ER/SR. This review addresses three important facets that control the SERCA2 activity in the heart. First, we focus on the alternative splicing of the SERCA2 messenger, which is strictly regulated in the developing heart. This splicing controls the formation of three SERCA2 splice variants with different enzymatic properties. Second, we will discuss the role and regulation of SERCA2a activity in the normal and failing heart. The two well‐studied Ca2+ affinity modulators phospholamban and sarcolipin control the activity of SERCA2a within a narrow window. An aberrantly high or low Ca2+ affinity is often observed in and may even trigger cardiac failure. Correcting SERCA2a activity might therefore constitute a therapeutic approach to improve the contractility of the failing heart. Finally, we address the controversies and unanswered questions of other putative regulators of the cardiac Ca2+ pump, such as sarcalumenin, HRC, S100A1, Bcl‐2, HAX‐1, calreticulin, calnexin, ERp57, IRS‐1, and −2.

A lipid switch unlocks Parkinson's disease- associated ATP13A2

Significance ATP13A2 is a lysosomal transporter that is genetically linked to an autosomal recessive variant of Parkinson’s disease and confers protection against α-synuclein toxicity in neurons. Here we show that an N-terminal hydrophobic domain of ATP13A2 specifically recognizes signaling lipids. Interactions with these signaling lipids enhance cytoprotection to mitochondrial stress. This study provides essential information for establishing the lysosomal function of ATP13A2 and suggests a therapeutic applicability in activating ATP13A2. ATP13A2 is a lysosomal P-type transport ATPase that has been implicated in Kufor–Rakeb syndrome and Parkinson’s disease (PD), providing protection against α-synuclein, Mn2+, and Zn2+ toxicity in various model systems. So far, the molecular function and regulation of ATP13A2 remains undetermined. Here, we demonstrate that ATP13A2 contains a unique N-terminal hydrophobic extension that lies on the cytosolic membrane surface of the lysosome, where it interacts with the lysosomal signaling lipids phosphatidic acid (PA) and phosphatidylinositol(3,5)bisphosphate [PI(3,5)P2]. We further demonstrate that ATP13A2 accumulates in an inactive autophosphorylated state and that PA and PI(3,5)P2 stimulate the autophosphorylation of ATP13A2. In a cellular model of PD, only catalytically active ATP13A2 offers cellular protection against rotenone-induced mitochondrial stress, which relies on the availability of PA and PI(3,5)P2. Thus, the N-terminal binding of PA and PI(3,5)P2 emerges as a key to unlock the activity of ATP13A2, which may offer a therapeutic strategy to activate ATP13A2 and thereby reduce α-synuclein toxicity or mitochondrial stress in PD or related disorders.