Calcitonin forms oligomeric pore-like structures in lipid membranes

Abstract

Calcitonin is a polypeptidic hormone involved in calcium metabolism in the bone. It belongs to the amyloid protein family, which is characterized by the common propensity to aggregate acquiring a beta-sheet conformation and include proteins associated with important neurodegenerative diseases. Here we show for the first time, to our knowledge, by transmission electron microscopy (TEM) that salmon-calcitonin (sCT) forms annular oligomers similar to those observed for beta-amyloid and alpha-sinuclein (Alzheimer's and Parkinson's diseases). We also investigated the interaction between sCT and model membranes, such as liposomes, with particular attention to the effect induced by lipid "rafts" made of cholesterol and G(M1). We observed, by TEM immunogold labeling of sCT, that protein binding is favored by the presence of rafts. In addition, we found by TEM that sCT oligomers inserted in the membrane have the characteristic pore-like morphology of the amyloid proteins. Circular dichroism experiments revealed an increase in beta-content in sCT secondary structure when the protein was reconstituted in rafts mimicking liposomes. Finally, we showed, by spectrofluorimetry experiments, that the presence of sCT allowed Ca(2+) entry in rafts mimicking liposomes loaded with the Ca(2+)-specific fluorophore Fluo-4. This demonstrates that sCT oligomers have ion-channel activity. Our results are in good agreement with recent electrophysiological studies reporting that sCT forms Ca(2+)-permeable ion channels in planar model membranes. It has been proposed that, beyond the well-known interaction of the monomer with the specific receptor, the formation of Ca(2+) channels due to sCT oligomers could represent an extra source of Ca(2+) entry in osteoblasts. Structural and functional data reported here support this hypothesis.

FIGURE 1

sCT annular oligomers imaged by negative contrast TEM. The inset shows the oligomer dimensional distribution (∼260 objects). The arrows indicate typical particles corresponding to each class.

FIGURE 2

sCT strongly binds to liposomes made of DPPC/cholesterol/G_M1 mimicking the occurrence of lipid rafts in the lipid membranes. Immunogold labeling TEM, performed with anti-mouse IgG 5-nm gold conjugate, clearly reveals that sCT is localized at the liposome surface. Conversely, only a few gold particles have been found around liposomes made of plain DPPC (26).

FIGURE 3

Rafts containing liposomes (gray islands) opened on amorphous carbon substrate for TEM in the presence of sCT (A). Isolated annular structures, similar to those observed in Fig. 1, are clearly detectable as “doughnuts” in the dark background (high magnification in B and C). More interestingly, pore-like structures are clearly observable in liposomes (high magnification in D and E). Such features are quite repetitive and characterized by a hexagonal symmetry.

FIGURE 4

CD spectra of sCT reconstituted in two different types of liposomes: plain DPPC (dashed line) and DPPC/cholesterol/G_M1 (continuous line). Liposomes containing cholesterol/G_M1 mimic the occurrence of lipid rafts. The deconvolution of the CD curves gives (Random = 56%; Turn = 15%; α-helix = 5%; β-structures = 23%) in the absence and (Random = 46%; Turn = 19%; α-helix = 5%; β-structures = 30%) in the presence of rafts. As can be observed the random-coil content decreases, whereas β content rises in the presence of rafts.

FIGURE 5

Fluorescence spectra relative to Ca²⁺ influx experiments performed on liposomes loaded with Fluo-4. Liposomes made of DPPC/cholesterol/G_M1, mimicking the occurrence of lipid rafts (A, B), give a fluorescence signal after the addition of Ca²⁺ only when sCT is inserted in the membrane (B). Plain DPPC liposomes (C, D) do not change the fluorescence signal in the presence of sCT, demonstrating that Ca²⁺ enters liposomes only when both sCT and rafts are present.

FIGURE 6

Schematic representation of the model proposed by Volles et al. (15) to explain how αS induces calcium-dependent neurotoxicity in PD. Annular oligomers organize in a structure that interacts with the lipid bilayer forming harmful Ca²⁺-permeable channels.