Solid-state NMR and Functional Measurements Indicate That the Conserved Tyrosine Residues of Sarcolipin Are Involved Directly in the Inhibition of SERCA1

Abstract

The transmembrane protein sarcolipin regulates calcium storage in the sarcoplasmic reticulum of skeletal and cardiac muscle cells by modulating the activity of sarco(endo)plasmic reticulum Ca 2؉ -ATPases (SERCAs). The highly conserved C-terminal region ( 27 RSYQY-COOH) of sarcolipin helps to target the protein to the sarcoplasmic reticulum membrane and may also participate in the regulatory interaction between sarcolipin and SERCA. Here we used solid-state NMR measurements of local protein dynamics to illuminate the direct interaction between the Tyr 29 and Tyr 31 side groups of sarcolipin and skeletal muscle Ca 2؉ -ATPase (SERCA1a) embedded in dioleoylphosphatidylcholine bilayers. Further solidstate NMR experiments together with functional measurements on SERCA1a in the presence of NAc-RSYQY, a peptide representing the conserved region of sarcolipin, suggest that the peptide binds to the same site as the parent protein at the luminal face of SERCA1a, where it reduces V max for calcium transport and inhibits ATP hydrolysis with an IC 50 of ϳ200 M. The inhibitory effect of NAc-RSYQY is remarkably sequence-specific, with the native aromatic residues being essential for optimal inhibitory activity. This combination of physical and functional measurements highlights the importance of aromatic and polar residues in the C-terminal region of sarcolipin for regulating calcium cycling and muscle contractility.Normal physiological control of muscle contractility depends on the strict regulation of intracellular calcium cycling (1). Muscle relaxation is coupled to the reuptake of Ca 2ϩ by the sarcoplasmic reticulum (SR), 2 driven by the sarco(endo)plasmic reticulum Ca 2ϩ -ATPase (SERCA), a 110-kDa membraneembedded ion pump that maintains a 10,000-fold Ca 2ϩ concentration gradient across the SR membrane (2). The cardiac isoform of SERCA (SERCA2a) associates reversibly with the pentameric transmembrane protein phospholamban (PLB) in the SR membrane resulting in a transitory inhibition of calcium transport into the SR, which is essential for efficient muscle contraction. Inhibition of SERCA2a is relieved during muscle relaxation as a result of phosphorylation of PLB by cAMP-dependent protein kinases in response to ␤-adrenergic stimulation.Sarcolipin (SLN) is a 31-amino acid PLB homologue that co-expresses and co-localizes with the fast twitch skeletal muscle (SERCA1a) isoform of Ca 2ϩ -ATPase and, to a lesser extent, with SERCA2a in cardiac cells (3-5). SLN causes a decrease in the apparent affinity of SERCA enzymes for calcium, but its effect on V max is less clear, with some studies noting an inhibitory effect and others showing a stimulatory response (6, 7). Expression of SLN in cardiac tissue is low in humans and predominates in the atria, whereas PLB levels are higher and localized in the ventricles, but there is mounting evidence that SLN plays a role in regulating calcium cycling in healthy and compromised heart muscle (10 -13). Co-expression of SERCA2a with SLN and PLB enhances the inhibition of calcium transport by S...

The Ca2+ Pumps of the Endoplasmic Reticulum and Golgi Apparatus

“…The aromatic residues of the highly conserved luminal extension RSYQYof SLN are functionally relevant (Odermatt et al 1998) and would interact with aromatic residues on the face of luminal loop L1-2 of SERCA ( possibly with the side chains of F73, W77, F88 or F92), opposite to that which constitutes the luminal interaction site of the 2b-tail (Fig. 2) (Hughes et al 2007). TM1 undergoes a strong upward movement during the enzymatic cycle, which might be affected by this interaction.…”

“…This would explain the super-inhibitory properties of the PLN-SLN heterodimers observed in vitro (Asahi et al 2002). Given the functional importance of the cytosolic domain of PLN and luminal extension of SLN, an additional stabilization of the complex might arise from their combined interaction with the pump (Hughes et al 2007). So far, there is no clear evidence for this superinhibition under physiological circumstances in the atria of the heart where both SERCA regulators are found (Bhupathy et al 2007;Periasamy et al 2008;Vandecaetsbeek et al 2009a).…”

Regulation of the Sarcoplasmic Reticulum Calcium Pump by Divergent Phospholamban Isoforms in Zebrafish

“…Because this could suggest convergent evolution of SLN and zfPLN, we were interested in the potential function of this unique domain. For SLN, the luminal domain plays a role in SR retention and SERCA inhibition (8,44,54), and this contrasts with what is known for PLN. PLN lacks a luminal domain, and SR retention occurs via the di-arginine motif in its cytoplasmic domain (55).…”

“…53 -PheHis-Gly-Met 57 ), which is lacking in all other known PLN sequences. Although SLN contains a luminal domain that contributes to SERCA inhibition (4,8,44), the key question was whether or not the luminal extension of zfPLN contributes to its inhibitory properties. We created two truncation variants of zfPLN, one with the last five C-terminal residues removed and another with the last six residues removed (designated zfPLN(Ϫ5) and zfPLN(Ϫ6), respectively; Fig.…”

“…Mutagenesis of the Zebrafish Luminal Domain-Aromatic residues play a key role in regulating SERCA from the luminal side of the SR membrane, as observed for the luminal domain of SLN (4,8,44) and the C-terminal tail of the ubiquitous SERCA2b splice variant (45,46). For this reason, we focused on the role of Phe 54 and His 55 in the luminal tail of zfPLN.…”