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-, and taurine in lipid bilayers and colocalizes with the Na+/K+-ATPase and the Na+/Ca2+-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 alpha-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 degrees +/- 2 degrees obtained by attenuated total reflection infrared spectroscopy. Thus, our data support the idea of ion channel formation by the PLM transmembrane domain.

Figure 1.

(A) (Top) Graph of the relative mobility R_f of the marker proteins in perfluoro-octaneoate (PFO)–PAGE in dependence of the logarithm of molecular mass. The arrow indicates the relative mobility of the phospholemman (PLM) peptide. (Bottom) PFO-PAGE gel showing marker proteins (left lane) and PLM (right lane). (B) Same as A for SDS-PAGE. The molecular mass of the marker proteins is 16.9 kDa, 14.4 kDa, 10.6 kDa, 8.2 kDa, 6.2 kDa, 3.5 kDa, and 2.5 kDa. The vertical bars indicate the standard error of the measurement.

Figure 2.

(A) Attenuated total reflection (ATR)–FTIR absorption spectra of the amide I/amide II region of lipid vesicle reconstituted PLM. The solid curve shows the spectrum obtained at a polarization parallel to the plane of incidence; the dashed curve shows the spectrum obtained at a polarization perpendicular to the plane of incidence. (B) The polarization-independent FTIR absorption spectrum after Fourier self-deconvolution (solid line). The dashed curve shows the individual component Gaussian curves used for curve-fitting the spectrum.

Figure 3.

Polarization-independent FTIR absorption spectra of lipid reconstituted PLM from H₂O (solid line) and after 9 h of incubation with D₂O saturated nitrogen gas (dashed line).

Figure 4.

Model of the PLM transmembrane domain, representing α-helices as cylinders. The light gray shading highlights the hydrophobic amino acid residues, while the hydrophilic residues are shaded in dark gray. The approximate position of the lipid bilayer is indicated. The top part of the figure represents the extracellular region, and the bottom part, the intracellular region. The N-terminal Asp (top) and C-terminal Arg residues of the transmembrane peptide oligomer are highlighted. This figure was created with VMD (Humphrey et al. 1996).