Interaction of heparins and dextran sulfates with a mesoscopic protein nanopore

Abstract

A mechanism of how polyanions influence the channel formed by Staphylococcus aureus alpha-hemolysin is described. We demonstrate that the probability of several types of polyanions to block the ion channel depends on the presence of divalent cations and the polyanion molecular weight and concentration. For heparins, a 10-fold increase in molecular weight decreases the half-maximal inhibitory concentration, IC(50), nearly 10(4)-fold. Dextran sulfates were less effective at blocking the channel. The polyanions are significantly more effective at reducing the conductance when added to the trans side of this channel. Lastly, the effectiveness of heparins on the channel conductance correlated with their influence on the zeta-potential of liposomes. A model that includes the binding of polyanions to the channel-membrane complex via Ca(2+)-bridges and the asymmetry of the channel structure describes the data adequately. Analysis of the single channel current noise of wild-type and site-directed mutant versions of alpha-hemolysin channels suggests that a single polyanion enters the pore due to electrostatic forces and physically blocks the ion conduction path. The results might be of interest for pharmacology, biomedicine, and research aiming to design mesoscopic pore blockers.

Figure 1

Representations of (A) the αHL channel, heparin, and DS used in this work, (B) the charge distribution at the αHL channel, and (C and D) the side-dependent heparin effect. (A) Cross section through the αHL channel (Protein Data Bank, 7AHL.pdb) embedded in membrane. Structure of the dodecameric heparin (Protein Data Bank, 1HPN.pdb; molecular mass of 3453.64 g/mol) and eventual structure of the hexadecameric DS (molecular mass of 5009.56 g/mol) were built with Chem3D (CambridgeSoft). (Abbreviations: Hep, heparin; DS, dextran sulfate.) The atoms are represented using the following color codes: O (which possesses a negative charge) in red, C in gray, H in white, N (which possesses a positive charge) in blue, and S in yellow. (Green triangles) Levels where the novel Cys and its charged derivatives are located in the channel structure. Scale bar is 2 nm. (B) The charge distribution was calculated using Coulomb calculation method (Swiss-PDBViewer, Ver. 3.7) assuming solvent ionic strength of 0.15 mol dm⁻³, equal to that of the 50 mM CaCl₂ solution mainly used in this study. The isopotential contouring values, equal to 1.8 kT/e, are shown (red, positive potentials; blue, negative potentials). (C and D) The current decays after application of 100 mV steps to multichannel bilayers in the presence of Hep6000 at the trans (C) and cis (D) compartments of the experimental chamber. (Red lines) Best-fit of a single exponential function. Control traces (no PA) are shown for comparison. Concentrations of Hep6000, voltage protocols, current, and timescales are given in the figure. Note that the current inhibition is not total. All other conditions for the experiment are described in Materials and Methods.

Figure 2

Influence of polyanions on αHL channel current reduction. αHL channel blockage as a function of heparin's concentration in the (A) cis or (B) trans compartment of the chamber. (Solid line) Best-fit of a one-site-binding equation. (Symbols: ○, HepAlb; ■, Hep; ▿, Hep6000; and ▴, Hep3000.) (C) The interrelation between molecular weight and IC₅₀ of heparin. (Solid line) First-order regression through the points. (Dashed lines) Extrapolations of the dependences to experimentally established values of IC₅₀ to HepAlb (x). (Arrow) Apparent molecular weight of heparin in HepAlb. The value r is a correlation coefficient. (D) The influence of DS (presented in the cis compartment of the chamber) on αHL channel blockage. Effect of the Hep is shown for comparison. (Solid line) Best-fit of a one-site-binding equation. (Symbols: ■, Hep; ▵, DS500; ●, DS10; and □, DS5.) Data are reported as means ± SD obtained in 5–7 independent experiments like those shown in Fig. 1, C and D.

Figure 3

Heparin effects at the single channel level. (A) Single αHL channel. The representative current trace was recorded at 140 mV. Hep3000 (390 μg/mL) was placed at the trans side. Recording was performed at 0.02-ms resolution. (Vertical arrow) Channel insertion. (Rectangle) A single short-blockage-event shown in higher time-resolution (inset). (Circle) Example of the trace used in current noise analysis. The respective all-point histograms are shown at the right, where Z denotes the current through unmodified lipid bilayer, and O and B correspond to the open and blocked channel levels, respectively. (B) Probability to find the channel in fully open state in the presence of Hep3000 (130 μM) at the cis or at the trans compartment. (Solid lines) Best-fit of a first-order exponential function. An e-fold change in the probability of being open was observed for change in every 24 mV. (C) Conductance histograms of the residual conductance of the channel under influence of Hep3000. The histograms were comprised from the observation of the approximately 100 blockage events shown in panel A. Bin width was 4 pS. (Solid line) Best-fit of a single normal distribution to the most probable conductance values for Hep3000 acting from the trans and the cis entrances of the αHL channel.

Figure 4

Influence of the strong positive or negative charge at the lumen of the channel close to the cis (A and B; I7C) or the trans (C and D; T129C) αHL channel entrance on the heparin effects. Multichannel lipid bilayers were used. Maximal value of the current, seen soon after potential shift from zero mV, was taken as 100 arbitrary units. Hep6000 was used at concentration of ∼4 μg/mL at the cis compartment (A and C) when voltage was switched from 0 to −100 mV. In experiments with 0–100 mV voltage switch, Hep6000 was used at a concentration of ∼0.4 μg/mL at the trans compartment (B and D). The traces labeled DTT are the Cys mutants kept in a reduced form. The reagents used for derivatization of cysteine (DTNB, MTSET) are given in the figure.

Figure 5

ζ-Potential of liposomes in the presence of growing concentrations of heparins and the correlation between equi-effective concentration of heparins against liposomes and αHL channels (inset). (Symbols: ○, HepAlb; ■, Hep; □, Hep6000; and ▵, Hep3000.) The value r is the correlation coefficient.

Figure 6

Relative effectiveness of heparins at the cis and the trans applications against their charge difference is presented in linear and semi-logarithmic (inset) plot. To build the plots, values of IC₅₀ were taken in nM. The ratio was defined as ${\text{IC}}_{\text{50}}^{\text{Hep3000}}/{\text{IC}}_{\text{50}}^{{\text{Hep}}_{\text{i}}}$. The number of charges per a heparin molecule, z, was taken equal to the number of sulfate groups. The effective charge difference was defined as Z_Hep3000–Z_Hepi. The correlation coefficient between experimental data and theory is >0.99.