1.A.50. The Phospholamban (Ca²⁺-channel and Ca²⁺-ATPase Regulator) (PLB) Family

Phospholamban (PLB) is the major phosphorylatable transmembrane protein of cardiac sarcoplasmic reticulum. It is 52 amino acyl residues long, and has been sequenced and characterized from mammals, the puffer fish, Tetraodon nigroviridis, and the chicken, Gallus gallus. Residues 1-31 (domains 1A (1-20) and 1B (21-31) are localized to the cytoplasm, while residues 32-52 (domain II) are predicted to span the membrane. It can be phosphorylated by protein kinases on residues 16 and 17. It assembles into a homopentameric complex in the native cardiac sarcoplasmic reticulum (SR) where it inhibits the activity of the P-type Ca²⁺ ATPase (TC #3.A.3) found in these membranes by decreasing its energetic efficiency. The pentameric (not the monomeric) PLB is necessary for the regulation of the Ca²⁺ ATPase and for myocardial contractility in vivo. PLB domain IA is the phosphorylation domain, and PLB domain IB interacts with the loop between TMSs 6 and 7 in the SR Ca²⁺-ATPase (Asahi et al., 2001). Binding to the ATPase causes structural changes in PLB (Hughes and Middleton, 2003). The conformational switch of phospholamban in calcium pump regulation has been examined when considering the interaction surfaces of these two membrane proteins (Zamoon et al. 2005). PLN may undergo allosteric activation upon encountering SERCA. The phospholamban pentamer alters the function of the sarcoplasmic reticulum calcium pump, SERCA (Glaves et al. 2019). There are alternative phospholamban-binding sites on the SERCA calcium transporter (Alford et al. 2020). Conformational changes within the cytosolic portion of phospholamban occur upon release of Ca-ATPase inhibition (Li et al. 2004). Small membrane proteins that are involved in Ca²⁺ transport and regulate cardiac and skeletal muscle contractility include phospholamban (PLN, 6 kDa), sarcolipin (SLN, 4 kDa), and DWORF (4 kDa) have been examined for their structures both free and in complex with SERCA. Phospholamban has been shown to form cation-selective channels in lipid bilayers, with Ca²⁺ being transported in preference to K⁺ (Kovacs et al., 1988). It spontaneously opens and closes, and the transmembrane region, residues 26-52, is sufficient for channel activity.  The structure of the phospholamban pentamer reveals a channel-like architecture in membranes (Oxenoid and Chou 2005). The putative regulatory portion of the protein, residues 2-25, do not form a channel. Possibly phospholamban regulates sarcoplasmic reticular Ca²⁺ flux by acting as a Ca²⁺ channel. However, channel activity is controversial (Becucci et al., 2009; Maffeo and Aksimentiev 2009). Heparin-derived oligosaccharides (HDOs) interact with the cytoplasmic domain of PLB and consequently stimulate SERCA activity (Hughes et al., 2010).  Motion of the transmembrane domain is restricted, but the cytoplasmic domain exhibits at least two distinct conformations (Nesmelov et al. 2007). Serine 16 phosphorylation induces an order-to-disorder transition in monomeric phospholamban (Metcalfe et al. 2005).

Phosphorylation of PLB abolishes its inhibitory effect on SERCA and therefore promotes Ca²⁺ transport into the SR lumen, enhancing cardiac relaxation. Phosphorylation occurs in response to β-adrenergic agonists. Pentamerization is believed to be mediated via the transmembrane domain of PLB, and phosphorylation may control the monomer-pentamer transition. Thus, PLB is a major regulator of the SR Ca²⁺ ATPase and of cardiac contractility, and phosphorylation may provide the primary mechanism for the control of these biochemical and physiological activities. Evidence suggests that one face of the PLB transmembrane helix interacts with helix M6 to cause inhibition. At saturating [Ca²⁺] and in the absence of PLB phosphorylation, binding of a single Ca²⁺ ion in the transport sites of SERCA rapidly shifts the equilibrium toward a noninhibited SERCA-PLB complex (Fernández-de Gortari and Espinoza-Fonseca 2018).  Missense variants in phospholamban and cardiac myosin binding protein have been identified in patients with a family history and clinical diagnosis of dilated cardiomyopathy (Armanious et al. 2024).  Pathological mutations in the phospholamban cytoplasmic region affect its topology and dynamics modulating the extent of SERCA inhibition (Weber et al. 2024).

PLB decreases the Ca²⁺ affinity of SERCA and attenuates contractile strength. cAMP-dependent phosphorylation of PLB reverses Ca²⁺-ATPase inhibition with powerful contractile effects. Akin et al. 2013 presented the crystal structure of the PLB-SERCA complex at 2.8 Å resolution in the absence of Ca²⁺. The structure shows PLB bound to a conformation of SERCA in which the Ca²⁺ binding sites are collapsed and devoid of divalent cations (E2-PLB). Relief of SERCA inhibition by PLB phosphorylation is due to an order-to-disorder transition in the cytoplasmic domain of PLB, which allows this domain to extend above the membrane surface and induce a structural change in the cytoplasmic domain of SERCA (Karim et al. 2006). Two kinds of motions of the helical domains can play functional roles. The population of conformations with relatively open interdomain angles, as well as large fluctuations of this coordinate in the bilayer, allows the N-terminal helix to come into contact with the PLB binding site on the calcium ATPase (Houndonougbo et al. 2005).

In lipid bilayers, PLN adopts a pinwheel topology with a narrow hydrophobic pore, which excludes ion transport. In the T state, the cytoplasmic amphipathic helices (domains Ia) are absorbed into the lipid bilayer with the transmembrane domains arranged in a left-handed coiled-coil configuration, crossing the bilayer with a tilt angle of approximately 11° with respect to the membrane normal (Verardi et al., 2011). The tilt angle difference between the monomer and pentamer is approximately 13°. Thus, both topology and function of PLN are shaped by the interactions with lipids. The cytoplasmic domain of PLB may act as a conformational switch, alternating between an orientation that lies across the membrane surface and an upright orientation that associates with the regulatory site of SERCA (Clayton et al. 2005).  Phospholamban inhibits the cardiac calcium pump by interrupting an allosteric activation pathway (Cleary et al. 2024).

Smeazzetto et al. 2017 evaluated the effects of phospholamban and sarcolipin on calcium translocation and ATP hydrolysis by SERCA. For pre-steady-state current measurements, proteoliposomes containing SERCA and phospholamban or sarcolipin were adsorbed to a solid-supported membrane and activated by substrate concentration jumps. Phospholamban altered ATP-dependent calcium translocation by SERCA within the first transport cycle, whereas sarcolipin did not. Using pre-steady-state charge (calcium) translocation and steady-state ATPase activity under various calcium and/or ATP concentrations, promoting particular conformational states of SERCA, phospholamban could establish an inhibitory interaction with multiple SERCA conformational states with distinct effects on SERCA's kinetic properties. Once a particular mode of association is engaged, it persists throughout the SERCA transport cycle for multiple turnover events. Thus, they system exhibits conformational memory in the interaction between SERCA and phospholamban (Smeazzetto et al. 2017).

Sarcolipin is a 31 aa protein expressed in cardiac and skeletal muscle. It has hydrophilic N- and C-termini flanking a hydrophobic putative TMS. It negatively regulates the sarcoplasmic reticulum (SR) Ca²⁺ ATPase (SERCA) which transports Ca²⁺ into the SR, the contraction-relaxation cycle of the heart (Babu et al., 2006). The rate of and amount of Ca²⁺ transported into the SR determines both the rate of muscle relaxation and the Ca²⁺ load available for the next cycle of contraction. Sarcolipin inhibits SERCA as does phospholamban (TC #1.A.50) which also functions as a Ca²⁺ channel (Babu et al., 2006). Sarcolipin reduces Ca²⁺ transport by the skeletal muscle sarcoplasmic reticulum Ca²⁺-ATPase and results in heat generation (Mall et al., 2006). Possibly the interaction of sarcolipin with the Ca²⁺-ATPase is important for thermogenesis. Conserved tyrosyl residues in sarcolipin are directly involved in the inhibition of SERCA (Hughes et al., 2007).

Sarcolipin is 73% identical, 86% similar to the C-terminus of a protein from Danio revio of 1066 aas termed protocadherin-1-like protein (XM_690233). This protein is 63% identical and 75% similar to human protocadherin-1 (Q08174; 1026 aas), but not in the C-terminal region where the former protein is similar to sarcolipin. Structural similarities between sarcolipin and phospholamban suggest that they are homologous. In fact, the transmembrane regions of these two proteins exhibit 40% identity and 95% similarity.

Sarcolipin (SLN) forms channels selective toward chloride and phosphate ions when incorporated in a bilayer lipid membrane. ATP increases conductivity, and the dependence of the conductivity on the ATP concentration satisfies the Michaelis-Menten equation, with an association constant of 0.1 μM. Phenylphosphonium ion and adenosine monophosphate exert inhibitory effects on membrane permeabilization to phosphate by ATP if they are added before ATP, but not if they are added after it (Becucci et al., 2009). Thus, SLN acts as an ATP-induced phosphate carrier. Sarcolipin (SLN) in mice  uncouples the calcium ATPase pump of the sarcoplasmic reticulum, resulting in heat production. Mice up-regulate expression of SLN in response to cold challenge. This thermoregulatory mechanism is characterized as non-shivering muscle-based thermogenesis (NST). A high ratio of sln transcripts to the CaATPase (serca1) transcripts suggests that the fish, the smalleye opah (Lampris incognitus) may utilize a futile calcium cycling NST mechanism in the dark red pectoral fin muscle to generate heat (Franck et al. 2019).

Another inhibitor of SERCA is a structurally similar 1 TMS peptide, myoregulin (Anderson et al. 2015). At present, it has not been shown to be homologous to Phospholamban and Sarcolipin.  However it inhibits SERCA in the same way, and their effects are counteracted by another small peptide, called DWORF (Dwarf ORF).  These two peptides are encoded by ORFs withing large RNA molecules not previously thought to encode proteins (Nelson et al. 2016).

Micropeptide regulators of SERCA form oligomers that may exhibit pore formation, but they bind to the pump as monomers (Singh et al. 2019). The same structural determinants that support oligomerization are also important for binding to SERCA. However, the unique oligomerization/SERCA-binding profile of DWORF is in harmony with its distinct role as a PLB-competing SERCA activator, in contrast to the inhibitory functions of the other SERCA-binding micropeptides (Singh et al. 2019).

As noted above, the activity of SERCA is regulated by phospholamban (PLN) and sarcolipin (SLN). SLN physically interacts with SERCA and differentially regulates contractility in skeletal and atrial muscle and is implicated in skeletal muscle thermogenesis. Wild-type SLN and a pair of mutants, Asn(4)-Ala and Thr(5)-Ala, yielded gain-of-function behavior comparable to what has been found for PLN (Glaves et al. 2020). Two-dimensional crystals of SERCA in the presence of wild-type SLN were examined by electron cryomicroscopy, revealing antiparallel dimer ribbons of SERCA, known as an assembly of calcium-free SERCA molecules induced by the addition of decavanadate. A projection map of the SERCA-SLN complex was determined to a resolution of 8.5 Å allowing the direct visualization of an SLN pentamer which interacted with TMS M3 of SERCA, although the interaction appeared to be indirect and mediated by an additional density consistent with an SLN monomer. This SERCA-SLN complex correlated with the ability of SLN to decrease the maximal activity of SERCA, which is distinct from the ability of PLN to increase the maximal activity of SLN (Glaves et al. 2020).

Phospholamban (PLN) directly controls the cardiac Ca²⁺-transport response to β-adrenergic stimulation, thus modulating cardiac output during the fight-or-flight response (Weber et al. 2021). In the sarcoplasmic reticulum (SR) membrane, PLN binds to SERCA, keeping this enzyme's function within a narrow physiological window. PLN phosphorylation by cAMP-dependent protein kinase A or an increase in Ca²⁺ concentration reverses the inhibitory effects. Phosphorylation of PLN's cytoplasmic regulatory domain disrupts 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 (Weber et al. 2021). Several studies suggested that PLB (PLN) can form a cationic selective channel, while others suggest that it can only store ions. Cao et al. 2022 reported the distribution of different membrane lipids and their effects on the structural dynamics and conductance properties of PLN pentamers of different systems. Cholesterol is highly enriched around the protein and stabilizes the structure of the PLN pentamer while the absence of cholesterol increases the flexibility of the protein backbone. The conductance properties of monovalent ions and water suggest that they cannot spontaneously permeate through the PLN pentamer channel pore, but the energy barrier is much lower in the absence of cholesterol, underlining the need to fully consider multiple lipid species when investigating small transmembrane protein oligomer ion-channel structure and conductance (Cao et al. 2022).

The sarco-endoplasmic reticulum calcium ATPase (SERCA) is responsible for maintaining calcium homeostasis in all eukaryotic cells by actively transporting calcium from the cytosol into the sarco-endoplasmic reticulum (SR/ER) lumen. Calcium is an important signaling ion, and the activity of SERCA is critical for a variety of cellular processes such as muscle contraction, neuronal activity, and energy metabolism. SERCA is regulated by several small transmembrane peptide subunits that are collectively known as the 'regulins'. Phospholamban (PLN) and sarcolipin (SLN) are the original and most extensively studied members of the regulin family. PLN and SLN inhibit the calcium transport properties of SERCA, and they are required for the proper functioning of cardiac and skeletal muscles, respectively. Myoregulin (MLN), dwarf open reading frame (DWORF), endoregulin (ELN), and another-regulin (ALN) are relatively newly discovered tissue-specific regulators of SERCA. Rathod et al. 2021 compared the functional properties of the regulin family of SERCA transmembrane peptide subunits and considered their regulatory mechanisms in the context of the physiological and pathophysiological roles of these peptides. They presented new functional data for human MLN, ELN, and ALN, demonstrating that they are inhibitors of SERCA with distinct functional consequences. Molecular modeling and molecular dynamics simulations of SERCA in complex with the transmembrane domains of MLN and ALN provide insight into how differential binding to the so-called inhibitory groove of SERCA-formed by TMSs M2, M6, and M9-can result in distinct functional outcomes (Rathod et al. 2021).

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