Effect of nanomolar concentrations of sodium dodecyl sulfate, a catalytic inductor of alpha-helices, on human calcitonin incorporation and channel formation in planar lipid membranes

Abstract

Human Calcitonin (hCt) is a peptide hormone which has a regulatory action in calcium-phosphorus metabolism. It is currently used as a therapeutic tool in bone pathologies such as osteoporosis and Paget's disease. However, due to its amphiphilic property tends to form a gelatinous solution in water which consists of fibrils that limits its therapeutic use. Here we show that sodium dodecyl sulfate (SDS), an anionic detergent able to induce and stabilize alpha-helices in polypeptides, at a monomeric concentration ranging between 0.26 mM-5 pM (all concentrations are below the CMC), increases the rate and number of hCt channel formation in planar lipid membranes, at both high and low hCt concentrations, with a maximum increase at a molecular hCt/SDS ratio of 1000:1. This effect could be interpreted as a counteraction to the fibrillation process of hCt molecules by removing molecules available for aggregation from the fluid; furthermore, this action, independently of channel formation in the cell membrane, could improve the peptide-receptor interaction. 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. The very low molecular ratio between SDS and peptide is suggestive of a possible catalytic action of SDS that could induce alpha-helices, the appropriate structures for interacting with the membrane. Moreover, in the experimental conditions investigated, the addition of SDS does not modify the membrane's electrical properties and most of the channel properties. This finding may contribute to the knowledge of environment-folding diseases due to protein and peptides.

FIGURE 1

Single-channel features of hCt in the absence and in the presence of SDS with associated histograms of the conductance fluctuations. (a) hCt 125 nM; (b) hCt 125 nM + SDS (0.26 mM); (c) hCt 24.5 nM; (d) hCt 24.5 nM + SDS (0.26 mM); (e) hCt 5 nM; and (f) hCt 5 nM + SDS (5 pM). Experiments were performed on a POPC/DOPG (85:15) membrane in the presence of hCt and of hCt + SDS added to the cis-side; the voltage was set to +150 mV, the aqueous phase contained 1M KCl (pH 7), and T = 22°C. Note the increase in channel occurrence (channels/minute) when SDS was present in the medium.

FIGURE 2

Single-channel features of hCt in the absence and in the presence of SDS at different concentrations with associated histograms of the conductance fluctuations. (a) hCt 125 nM; (b) hCt 125 nM + SDS (12.5 nM); (c) hCt 125 nM + SDS (1.25 nM); (d) hCt 125 nM + SDS (0.125 nM); (e) hCt 125 nM + SDS (0.0125 nM); (f) SDS (0.26 mM); and (g) SDS (0.125 nM). Experiments were performed on a POPC/DOPG (85:15) membrane in the presence of hCt and of hCt + SDS added to the cis-side; the voltage was set to +150 mV, the aqueous phase contained 1M KCl (pH 7), and T = 22°C.

FIGURE 3

Conductance-voltage relationship for hCt channels in the absence (▪) or presence of SDS (□). Experimental conditions: KCl 1M, hCt (49 nM) or hCt (49 nM) + SDS (0.26 mM) was present on the cis sides of the POPC/DOPG (85:15) membrane. The curves superimposed on the data are the results of the fit with the model: λ_c = Ae^(−KVm) + p, where A is the difference between the conductance at V_m = 0 and at V_m = membrane black (p); K is the constant correlated with the gating charge n (n = KRT/F). (▪) A = 0.234 ±0.004 (nS); p = 0.0067 (nS); K = 37.13 ± 0.59 (V⁻¹); R² = 0.988. (□) A = 0.29 ± 0.002 (nS); p = 0.0067 (nS); K = 38.3 ± 0.48(V⁻¹); R²= 0.99.