Secondary structure, orientation, and oligomerization of phospholemman, a cardiac transmembrane protein

Abstract

Human phospholemman (PLM) is a 72-residue protein, which is expressed at high density in the cardiac plasma membrane and in various other tissues. It forms ion channels selective for K + , Cl ÿ , and taurine in lipid bilayers and colocalizes with the Na + /K + -ATPase and the Na + /Ca 2+ -exchanger, which may suggest a role in the regulation of cell volume. Here we present the first structural data based on synthetic peptides representing the transmembrane domain of PLM. Perfluoro-octaneoate-PAGE of reconstituted proteoliposomes containing PLM reveals a tetrameric homo-oligomerization. Infrared spectroscopy of proteoliposomes shows that the PLM peptide is completely a-helical, even beyond the hydrophobic core residues. Hydrogen/deuterium exchange experiments reveal that a core of 20-22 residues is not accessible to water, thus embedded in the lipid membrane. The maximum helix tilt is 17°6 2°obtained by attenuated total reflection infrared spectroscopy. Thus, our data support the idea of ion channel formation by the PLM transmembrane domain.Keywords: secondary structure; oligomerization; membrane proteins; infrared spectroscopy; peptide; phospholemman Phospholemman (PLM) is a 72-residue protein that is found in the central nervous system and skeletal muscle and at high density in the cardiac sarcolemma-the plasma membrane that surrounds the heart muscle fiber-from which it was first purified (Palmer et al. 1991). It is phosphorylated by protein kinase A and C at different sites in the cytoplasmic domain (Palmer et al. 1991), which suggests that PLM may be an important control point in the function of heart cells. The biological importance of PLM is further emphasized by the fact that PLM exhibits a close similarity to a number of other newly identified membrane proteins, including CHIF, RIC, and MAT-8, that form a new superfamily of membrane proteins with a single transmembrane domain (Sweadner and Rael 2000).The function of PLM is not completely understood, yet recent evidence suggests that PLM may function as a regulator of the Na + /K + -ATPase (Silverman et al. 2005) and that it also colocalizes with the Na + /Ca 2+ -exchanger (Mirza et al. 2004). Electrical measurements of PLM in artificial lipid bilayers and oocytes show that PLM facilitates the membrane flux of ions (Chen et al. 1999) and taurine transport (Moorman et al. 1995). Based on these findings, it has been suggested that PLM has a function in the regulation of cell volume either by modulating of a swelling-activated signal transduction pathway or by directly facilitating osmolyte influx (Davis et al. 2004).To date, there are no structural data available about PLM based on physical measurements, however a solidstate NMR study of the related MAT-8 and CHIF proteins shows that they have an a-helical transmembrane domain (Franzin et al. 2004). On the basis of hydropathy analysis of the amino acid sequence, PLM is predicted to have a single transmembrane domain from residues 18-37 (Palmer et al. 1991). The C terminus is located intracellularly,...

Isoform Specificity of the Na/K-ATPase Association and Regulation by Phospholemman

“…This suggests that PLM may exist in the membrane as a homooligomer with three or more molecules. This is in agreement with structural data indicating that PLM may exist in the membrane as a tetramer (32). It is thus possible that PLM forms channels, as originally proposed by Moorman et al (40), although the physiological significance of such channels is unclear.…”

“…2B). The observed deviation from linearity supports the model that PLM can form homo-oligomers of at least three molecules (32). Effect of PLM Phosphorylation and Ouabain on FRET with NKA-␣1/NKA-␣2-We have shown previously that PLM phosphorylation by PKA and PKC enhances the NKA activity (10,11) and drastically reduces the FRET between canine NKA-␣1 and PLM (9).…”

Phospholemman Transmembrane Structure Reveals Potential Interactions with Na+/K+-ATPase

“…The symmetric amide I absorption band centered at ϳ1658 cm Ϫ1 revealed that in all cases, the peptide adopted a predominantly ␣-helical conformation (33), confirming earlier observations (19). The absorption band of the labeled sites centered at 1594 cm…”

“…3) is a right-handed ␣-helical bundle with a crossing angle close to 0°, whereas local helix tilt angles derived from the IR data vary from 2.5 to 10.1°, depending on the site in question, resulting in an average tilt angle of 7.3°. This is in accordance with previous conventional ATR-FTIR experiments, which indicated an average tilt angle between 0°and 17° (19), if the sample order is not taken into account.…”

“…The three-dimensional structure of PLM is not known, but on the basis of hydropathy analysis of the amino acid sequence, a transmembrane domain from residue 18 to 37 has been predicted (2) and confirmed later by attenuated total reflection (ATR) FTIR spectroscopy (19). The C terminus is located intracellularly, resistant to protease digestion, whereas the N terminus is located extracellularly, as shown by antibody labeling (20).…”