A New Class Of Peptide-Forming Channel: Calcitonins

Calcitonin is a small peptide hormone present in all vertebrates with an important effect on Ca2+ metabolism. It exerts its main action by inhibiting osteoclast-mediated bone resorption and inducing calcium regulation through an interplay of clearance through the kidney and intestine. This action is responsible for its widespread clinical use in the treatment of bone disorders, including Paget's disease, osteoporosis, hypercalcemia of malignancy, and musculoskeletal pain. Unfortunately, this peptide (at least the human calcitonin, or hCt) shows a high tendency to aggregate, resulting in fibrillation that impairs its use as a drug for the above pathologies. This could explain why other calcitonins with a lower tendency to fibrillate, in particular salmon and eel calcitonin (sCt, eCt), are currently in use.The mechanism of fibrillation is common to other peptides such as insulin, prion protein, Alzheimer A beta, and the alpha-synuclein responsible for Parkinson's disease, is far from being well understood despite the many investigations which have been undertaken. Thus, if the mechanism underlying the fibrillation of one peptide could be resolved, this may be extrapolated to the other fibrillating peptides, thus shedding light on the "channel pathologies'' which are presumably linked by a common denominator.The technique used to study the fibrillation mechanism is performed both in solution and on the membrane surface. In fact, it has been found that membrane phospholipids can play an important role either inducing or counteracting aggregation by incorporating peptides into the membrane.In the past few years, we have been performing systematic studies using model Planar Lipid Membranes (PLM) in order to:gather evidence of calcitonin incorporation into the membrane and channel formation under different experimental conditions (i.e., membrane applied voltage, bathing conditions);test the influence on hCt channel properties of varying the PLM composition, or the pH of the medium, and of inducing and stabilizing alpha-helices by means of an anionic detergent;understand the role of glycosylation at a different position in eCt during peptide incorporation and channel formation.The results obtained can be summarized as follows:on the ability to form channels: hCt and sCt are able to form channels when incorporated into DOPC/DOPG (molar ratio 85:15) model membranes. Moreover, the average single-channel conductance decreases as a function of the membrane applied voltage (from similar to 0.2/0.5 nS at +/- 10 mV for hCt and sCt, respectively, to similar to 0.014 nS at +/- 150 mV for both) and has different values under different bathing conditions (higher for NaCl, CaCl2, KCH3COO than for KCl). The permeability for cations/anions is almost identical. Finally eel, porcine, and carbocalcitonin also form channels, with a mean conductance higher than that of hCt and sCt.on the way in which the acid-base conditions in the bathing medium affect the hCt channels: At pH 7, hCt is able to interact and forms channels with negatively charged dioleoyl-phosphatidyl-glycerol (DOPG) bilayers and with zwitterionic palmitoyl-oleoyl-phosphatidylcholine (POPC) bilayers containing 15% negatively charged DOPG, but not with POPC bilayers. At low pH (4.4 and 3.8), the conformational variation of the peptide enables it to insert into POPC and POPC:DOPG but not into DOPG bilayers.on the effect of detergent on the hCt channels: Sodium dodecyl sulfate (SDS) an anionic detergent able to induce and stabilize alpha-helices of polypeptides, at concentrations ranging between 0.26 mM and 5 pM [all concentrations are below the critical micelle concentration (CMC)] increases the rate and number of hCt channel formation in PLM at both high and low hCt concentration, with a maximum increase at a molecular hCt/SDS ratio of 1000:1. The action of SDS could be attributable to the strength of the sulfate negative charge and the hydrophobic chain; in fact a similar effect was obtained with lauryl sarcosine and not with a neutral detergent such as n-dodecyl-beta-D-maltoside.on the glycosylation at different positions in eCt: eCt glycosylation at different positions (Ct3-GlcNAc, Ct14-GlcNAc, Ct20-GlcNAc, and Ct26-GlcNAc) preserves the molecular structure and slightly changes the energy of incorporation and channel formation into POPC: DOPG (85:15 w/w). The voltage needed to form channels decreases as the attached carbohydrate moves toward the C-terminal (eCt = Ct3-GlcNAc > Ct14-GlcNAc = Ct20-GlcNAc > Ct26-GlcNAc). Interestingly, all the Cts tested maintain the characteristic voltage-conductance dependence found for other Cts; the only channel property modified being ion selectivity that shifts toward anion selectivity (eCt 0.97, Ct3-GlcNAc = 0.49, Ct14-GlcNAc = 0.41, Ct20-GlcNAc = 0.36, and Ct26-GlcNAc = 0.47).