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).
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 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).
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).
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).
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).
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.
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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.
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 atwo peptides are encoded by ORFs withing large RNA molecules not previously thought to encode proteins (Nelson et al. 2016).
Phospholamban (PLB) pentameric Ca2+/K+ channel (Kovacs et al., 1988; Smeazzetto et al. 2013; Smeazzetto et al. 2014). In spite of extensive experimental evidence, suggesting a pore size of 2.2 Å, the conclusion of ion channel activity for phospholamban has been questioned (Maffeo and Aksimentiev 2009). Phosphorylation by protein kinase A and dephosphorylation by protein phosphatase 1 modulate the inhibitory activity of phospholamban (PLN), the endogenous regulator of the sarco(endo)plasmic reticulum calcium Ca2+ ATPase (SERCA). This cyclic mechanism constitutes the driving force for calcium reuptake from the cytoplasm into the myocite lumen, regulating cardiac contractility. PLN undergoes a conformational transition between a relaxed (R) and tense (T) state, an equilibrium perturbed by the addition of SERCA. Phosphoryl transfer to Ser16 induces a conformational switch to the R state. The binding affinity of PLN to SERCA is not affected ((Kd ~ 60 microM). However, the binding surface and dynamics in domain Ib (residues 22-31) change substantially upon phosphorylation. Since PLN can be singly or doubly phosphorylated at Ser16 and Thr17, these sites may remotely control the conformation of domain Ib (Traaseth et al. 2006). Phospholamban interests with SERCA with conformational memory (Smeazzetto et al. 2017).
PLB of Homo sapiens (P26678)
Cardiac phospholamban-like protein of 131 aas and 1 TMS.
Phospholamban of Scleropages formosus
Cardiac phospholamban of 55 aas and 1 TMS.
Phospholamban of Esox lucius (northern pike)
Sarcolipin (SLN) anion pore-forming protein of 31 aas and 1 TMS, with selectivity for Cl- and H2PO4-. Oligomeric interactions of sarcolipin and the Ca-ATPase have been documented (Autry et al., 2011). Sarcolipin, but not phospholamban, promotes uncoupling of the SERCA pump (3.A.3.2.7; Sahoo et al. 2013). Forms a pentameric pore that can transport water, Na+, Ca2+ and Cl-. Leu21 serves as the gate (Cao et al. 2015). In the channel, water molecules near the Leu21 pore demonstrated a clear hydrated-dehydrated transition (Cao et al. 2016). Small ankyrin 1 (sAnk1; TC#8.A.28.1.2) and SLN interact with each other in their transmembrane domains to regulate SERCA (TC# 3.A.3.2.7) (Desmond et al. 2017).
SLN of Homo sapiens (O00631)
sarcolipin-like protein of 32 aas and 1 TMS.
Sarcolipin of Esox lucius (northern pike)
Sarcolipin-like protein (SLN) of 31 aas and 1 TMS
SLN of Ovis aries (Sheep)
Myoregulin of 46 aas (Anderson et al. 2015).
Myoregulin of Homo sapiens
Myoregulin of 43 aas
Myoregulin of Echinops telfairi
Myoregulin of 105 aas
Myoregulin of Sarcophilus harrisii (Tasmanian devil) (Sarcophilus laniarius)
DWORF of 34 aas; synthetic construct (Nelson et al. 2016). Counteracts the inhibitory effects of single transmembrane peptides, phospholamban (TC# 1.A.50.1), sarcolipin (1.A.50.2) and myoregulin (1.A.50.3), on SERCA (TC# 3.A.3.2). Homology with the inhibitory peptides has not been established although all of these peptides have about the same size with a single C-terminal TMS.
DWORF, made synthetically, probably copied from DWORF of Mus musculus
Endoregulin, ELN, also called small integral membrane protein-6, SMIM6, is of 62 aas and 1 TMS. This protein and the other members of the phospholamban family have been designated "micropeptides". Micropeptides function as regulators of calcium-dependent signaling in muscle. The sarco/endoplasmic reticulum Ca2+ ATPase (SERCA TC# 3.A.3.2.7), is the membrane pump that promotes muscle relaxation by taking up Ca2+ into the sarcoplasmic reticulum. It is directly inhibited by three known muscle-specific micropeptides: myoregulin (MLN), phospholamban (PLN) and sarcolipin (SLN). In non muscle cells, there are two other such micopeptides, endoregulin (ELN) and "another-regulin (ALN) (Anderson et al. 2016). Endoregulin is also known as "small integral membrane protein-6" (SMIM6) while ALN is also known as Protein C4 orf3 (C4orf3). These proteins share key amino acids with their muscle-specific counterparts and function as direct inhibitors of SERCA pump activity. The distribution of transcripts encoding ELN and ALN mirrored that of SERCA isoform-encoding transcripts in nonmuscle cell types. Thus, these two proteins are additional members of the SERCA-inhibitory micropeptide family, revealing a conserved mechanism for the control of intracellular Ca2+ dynamics in both muscle and nonmuscle cell types (Anderson et al. 2016).
Endoregulin of Homo sapiens
ELN homologue of 78 aas and 1 TMS.
ELN homologue of Nothobranchius furzeri
ELN homologue of 75 aas and 1 TMS.
ELN of Larimichthys crocea (large yellow croaker)
Bacterial ELN homologue of unknown function with 101 aas and 1 TMS
ELN homologue of Desulfobacteraceae bacterium
ELN homologue of 85 aas and 1 TMS
ELN homologue of Thermotoga sp.
"Another-regulin", ALN, of 66 aas and 1 TMS. Also called Protein C4orf3. This protein and the other members of the phospholamban family have been designated "micropeptides". Micropeptides function as regulators of calcium-dependent signaling in muscle. The sarco/endoplasmic reticulum Ca2+ ATPase (SERCA, TC# 3.A.3.2.7), is the membrane pump that promotes muscle relaxation by taking up Ca2+ into the sarcoplasmic reticulum. It is directly inhibited by three known muscle-specific micropeptides: myoregulin (MLN), phospholamban (PLN) and sarcolipin (SLN). In non muscle cells, there are two other such micopeptides, endoregulin (ELN) and "another-regulin" (ALN) (Anderson et al. 2016). These proteins share key amino acids with their muscle-specific counterparts and function as direct inhibitors of SERCA pump activity. The distribution of transcripts encoding ELN and ALN mirror that of SERCA isoform-encoding transcripts in nonmuscle cell types. Thus, these two proteins are additional members of the SERCA-inhibitory micropeptide family, revealing a conserved mechanism for the control of intracellular Ca2+ dynamics in both muscle and nonmuscle cell types (Anderson et al. 2016).
ALN in Homo sapiens
Uncharacterized protein of 93 aas and 1 TMS.
UP of Larimichthys crocea (large yellow croaker)
Uncharacterized protein of 104 aas and 1 TMS
UP of Xenopus laevis (African clawed frog)
Uncharacterized C4orf3 homologue of77 aas and 1 TMS
UP of Monodelphis domestica (Gray short-tailed opossum)