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 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.
Diociaiuti
Marco
M
Dipartimento di Tecnologie e Salute, Istituto Superiore di Sanità, 299-00161 Rome, Italy. marco.diociaiuti@iss.it
Polzi
Laura Zanetti
LZ
Valvo
Luisa
L
Malchiodi-Albedi
Fiorella
F
Bombelli
Cecilia
C
Gaudiano
Maria Cristina
MC
D000814:Aniline Compounds; D002116:Calcitonin / Q000737:chemistry*; D002118:Calcium / Q000737:chemistry; D015220:Calcium Channels / Q000737:chemistry; D002784:Cholesterol / Q000737:chemistry*; D002942:Circular Dichroism; D005456:Fluorescent Dyes; D008051:Lipid Bilayers / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry; D021962:Membrane Microdomains / Q000737:chemistry*; D046529:Microscopy, Electron, Transmission; D016062:Porosity; D017433:Protein Structure, Secondary; D014966:Xanthenes
D000814:Aniline Compounds; D015220:Calcium Channels; C409648:Fluo 4; D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D008081:Liposomes; D014966:Xanthenes; C028815:salmon calcitonin; D002116:Calcitonin; D002784:Cholesterol; D002118:Calcium