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.
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).
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.
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).