Conductance and ion selectivity of a mesoscopic protein nanopore probed with cysteine scanning mutagenesis

Abstract

Nanometer-scale proteinaceous pores are the basis of ion and macromolecular transport in cells and organelles. Recent studies suggest that ion channels and synthetic nanopores may prove useful in biotechnological applications. To better understand the structure-function relationship of nanopores, we are studying the ion-conducting properties of channels formed by wild-type and genetically engineered versions of Staphylococcus aureus alpha-hemolysin (alphaHL) reconstituted into planar lipid bilayer membranes. Specifically, we measured the ion selectivities and current-voltage relationships of channels formed with 24 different alphaHL point cysteine mutants before and after derivatizing the cysteines with positively and negatively charged sulfhydryl-specific reagents. Novel negative charges convert the selectivity of the channel from weakly anionic to strongly cationic, and new positive charges increase the anionic selectivity. However, the extent of these changes depends on the channel radius at the position of the novel charge (predominantly affects ion selectivity) or on the location of these charges along the longitudinal axis of the channel (mainly alters the conductance-voltage curve). The results suggest that the net charge of the pore wall is responsible for cation-anion selectivity of the alphaHL channel and that the charge at the pore entrances is the main factor that determines the shape of the conductance-voltage curves.

FIGURE 1

Molecular models of αHL. (A) Schematic illustration of sagittal section through the heptameric αHL pore inserted in lipid bilayer. The cartoon shows the relative sizes of the pore, the positions of the main constriction, cap, and stem regions of the channel in the lipid bilayer. (B) Ribbon representation of the αHL pore. The positions some of amino acids, which are the loci for the substitutions described herein are shown in the space-filled representation. The longitudinal section and the ribbon representation of αHL channel (Protein Data Bank code 7AHL.pdb) were visualized with CS Chem3D Pro (CambridgeSoft). The final representations were produced with Adobe Photoshop.

FIGURE 2

Voltage dependence of wild-type αHL channel conductance in the presence of different potassium chloride concentrations. The relative single-channel conductances, which are normalized at −200 mV transmembrane voltage, decrease as the applied potential is increased. In addition, an increase in the electrolyte concentration decreases the effect of voltage on the conductance. The electrolyte solutions are buffered with 5 mM Tris and adjusted to pH 7.5 with citric acid. Each point presents the mean value ± SD of at least seven separate experiments.

FIGURE 3

Conductance histogram of single channels formed by the genetically engineered protein αHL G134C. The inset illustrates a typical current recording of single channels spontaneously formed by G134C αHL monomer added to the cis compartment (final concentration ∼5 ng/ml). Current records were not analyzed if any of the open channels closed temporarily (i.e., gated). The histogram is comprised of the conductance values of 210 channel formation events (5–15 channels per membrane) and the bin width is 6 pS. The solid line indicates the best fit of a single normal distribution to the most probable conductance values near 120 pS. The applied potential was −40 mV. All other conditions for the experiment are described in the Methods section.

FIGURE 4

Steady-state conductance-voltage relationships of single channels formed by wild-type and novel point cysteine mutant versions of αHL. Each G-V data set represents the averages of 3–5 independent experiments. The standard deviations, which did not exceed 10% of the conductance values, are omitted for clarity.

FIGURE 5

Effect of single cysteine substitutions on the αHL channel rectification. The dashed line represents the ratio of the conductance value at −100 mV to that at +100 mV (G₋₁₀₀/G₊₁₀₀) for the wild-type αHL channel. The abscissa denotes the distance between each novel cysteine side chain and trans channel opening. The sequences of mutants from left to right are (A) T129C, K131C, D127C, G133C, L135C, G137C, N121C, N139C, S141C, and G143C, and (B) G130C, D128C, I132C, G126C, G134C, V124C, I136C, G122C, A138C, F120C, V140C, and Y118C. The conductance rectification ratio of the channels formed by D108C and I7C (in the αHL channel cap region at 5.74 nm and 8.50 nm from the trans opening) are 1.81 ± 0.07 and 1.55 ± 0.06, respectively. Each point represents the mean value of G₋₁₀₀/G₊₁₀₀ obtained in 3–5 separate experiments.

FIGURE 6

Effect of single cysteine substitutions on the reversal potential of αHL channels. The reversal potential was measured in the presence of a threefold KCl concentration gradient (300 mM cis, 100 mM trans) in the presence of 1 mM DTT and 30 mM Tris. The dashed line represents the value of the reversal potential for channels formed by wild-type αHL. The sequences of mutants from left to right are: odd, T129C, K131C, D127C, G133C, L135C, G137C, N121C, N139C, S141C, and G143C; and even, G130C, D128C, I132C, G126C, G134C, V124C, I136C, G122C, A138C, F120C, V140C, and Y118C. The reversal potentials of channels formed by D108C and I7C are 6.9 ± 0.9 and 6.4 ± 0.3, respectively. Each point represents the mean value of V_rev obtained in 5–7 separate experiments.

FIGURE 7

Single-channel recordings of G143C-αHL channels before and after the addition of the sulfhydryl reagents DTNB and MTSET. G143C was added to the cis solution at a concentration of ∼0.2 ng/ml. After a single channel appeared, DTNB (A) or MTSET (B) was added at a concentration of 1.5 mM and 1 mM to the cis and trans compartments, respectively. After a few seconds, the channel conductance changed in a stepwise manner (A) or smoothly (B), indicative of its reaction with reagents. Several clearly resolved steps can be seen in A. Solutions in both compartments of the chamber contained KCl (1000 mM (A) or 100 mM (B)), 0.5 mM DTT, 1 mM EDTA, 30 mM Tris-HCl, pH 7.5. The pulse protocol is shown in the top part of the figure. In each case, the sign of applied potential was chosen to make the greatest possible reagent-induced change in the channel conductance.

FIGURE 8

Conductance-voltage relationships of channels formed by the odd-numbered single cysteine αHL mutants derivatized by MTSET (A) and MTSES (B). The SH reagents are added to both bilayer chamber compartments. Each curve represents the mean of three independent experiments. The standard deviation values (not shown) do not exceed 10%.

FIGURE 9

Effect of sulfhydryl reagents on the αHL cysteine mutant channel rectification. Each point represents the mean rectification ratio, G_{−100 mV}/G_{+100 mV}, obtained in 3–5 separate experiments. The dashed line represents the value of G₋₁₀₀/G₊₁₀₀ for the wild-type αHL channel. The sequence of mutants from left to right is T129C, G130C, K131C, D127C, G133C, L135C, G137C, N121C, N139C, S141C, and G143C.

FIGURE 10

Effects of sulfhydryl reagents on the reversal potential of channels formed by single cysteine αHL mutants. (A) The membrane is bathed by 100 mM and 300 mM KCl aqueous solution on the trans and cis sides, respectively. (B) 3000 mM/1000 mM (cis/trans) KCl gradient is used. The maximum and minimum values of P_K/P_Cl were 3.0 and 0.35 and were caused by MTSES and MTSET, respectively. The sequence of mutants from left to right is T129C, G130C, K131C, D127C, G133C, L135C, G137C, N121C, N139C, S141C, and G143C. Except for G130C, all other channels formed by even-numbered point cysteine mutants, including D108C, were insensitive to the SH reagents. The dashed line represents the value of the reversal potential for the channel formed by wild-type αHL. Each point represents the mean value (± SD) of V_rev obtained in 5–7 independent experiments.

FIGURE 11

Interrelation between the variation of the reversal potential of ion channels formed by cysteine-substituted derivatized αHL mutants and the radius of the wild-type channel. (A) The variation of the reversal potential (○, ▵) of ion channels built by cysteine-substituted derivatized αHL mutants and the radius (▪) of the channel with the distance from the trans opening . ▵, MTSES treatment; ○, MTSET treatment. The data are the absolute value of the difference in V_rev of ion channels before and after derivatization in the presence of a 3000 mM/1000 mM (cis/trans) KCl gradient. Each point represents the mean values obtained in 5–7 separate experiments. The sequence of mutants from left to right is as follows: T129C, K131C, D127C, G133C, L135C, G137C, N121C, N139C, S141C, G143C. The radii were determined from the α-carbon of each odd amino acid residue in the stem region using the αHL channel crystallographic structure (7AHL.pdb), the Swiss-Pdb Viewer version 3.7 (49), and CS Chem3D Pro (CambridgeSoft). (B) The correlation between the variation of the reversal potential and the radii of channels formed by point cysteine mutants.