Calcitonin forms oligomeric pore-like structures in lipid membranes

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 spectro fluorimetry experiments, that the presence of sCT allowed Ca2+ entry in rafts mimicking liposomes loaded with the Ca2+-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 Ca2+-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 Ca2+ channels due to sCT oligomers could represent an extra source of Ca2+ entry in osteoblasts. Structural and functional data reported here support this hypothesis.