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Originally published as Biophys J. BioFAST on August 5, 2005.
doi:10.1529/biophysj.105.066472
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Biophysical Journal 89:3059-3070 (2005)
© 2005 The Biophysical Society

Conductance and Ion Selectivity of a Mesoscopic Protein Nanopore Probed with Cysteine Scanning Mutagenesis

Petr G. Merzlyak *, Maria-Fatima P. Capistrano {dagger}, Angela Valeva {ddagger}, John J. Kasianowicz § and Oleg V. Krasilnikov *

* Laboratory of Membrane Biophysics, Department of Biophysics and Radiobiology, Universidade Federal de Pernambuco, Recife, Pernambuco, Brazil; {dagger} Department of Biophysics and Pharmacology, Federal University of Rio Grande de Norte, Natal, Rio Grande de Norte, Brazil; {ddagger} Institute of Medical Microbiology and Hygiene, University of Mainz, Germany; and § National Institute of Standards and Technology, Electronics and Electrical Engineering Laboratory, Semiconductor Electronics Division, Gaithersburg, Maryland 20899-8120

Correspondence: Address reprint requests to Dr. Oleg V. Krasilnikov, Universidade Federal de Pernambuco, Centro de Ciências Biológicas, Depto. de Biofísica e Radiobiologia, Av. prof. Moraes Rego, S/N, Cidade Universitária, Recife, Pernambuco, Brasil, CEP 50670-901. Tel.: 55-81-2126-8535; Fax: 55-81-2126-8560; E-mail: kras{at}ufpe.br.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 SUMMARY AND CONCLUSIONS
 ACKNOWLEDGEMENTS
 REFERENCES
 
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 ({alpha}HL) reconstituted into planar lipid bilayer membranes. Specifically, we measured the ion selectivities and current-voltage relationships of channels formed with 24 different {alpha}HL 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 (predominately 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 {alpha}HL channel and that the charge at the pore entrances is the main factor that determines the shape of the conductance-voltage curves.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 SUMMARY AND CONCLUSIONS
 ACKNOWLEDGEMENTS
 REFERENCES
 
By virtue of their action in nerve, muscle, and other tissues (1Go), protein ion channels are of key importance in biology. Recent work suggests that channels (2Go–11Go) and solid-state analogs (12Go–15Go) might prove useful in technological applications, which include rapid DNA sequencing and the simultaneous detection of multiple analytes. However, more insight into how channels catalyze ion and macromolecule transport is needed to aid the rational design of nanopore-based sensors.

Ion channels are an ideal model system for the study of nanopores because they self-assemble, and their structures can be altered using molecular biological techniques. For example, site-directed mutagenesis can be used to change the number and location of fixed charges inside the channel and/or near the pore entrances. The effects of such changes to the channel structure are generally reflected in the ion selectivity and the shape of the I-V relationship (16Go–20Go). To learn more about how charged amino acid side chains control the properties of ion channels, we studied the conducting properties of the channel formed by the bacterial exotoxin Staphylococcus aureus {alpha}-hemolysin ({alpha}HL) (21Go) and its genetically engineered mutants.

The {alpha}HL channel forms spontaneously from seven identical monomers (22Go–24Go), each with a molecular mass of 33.1 kDa. The crystal structure of the {alpha}HL channel was determined nearly a decade ago (23Go). The channel, illustrated in (Fig. 1 A) is ~10 nm long. It is comprised of a large cap domain and a much less massive stem region. The stem, which spans the lipid bilayer, is an almost cylindrical 14-stranded ß-barrel with an average diameter of ~2.6 nm between {alpha}-carbons on opposite sides of the pore (23Go,25Go,26Go). Both ends of the stem are decorated with rings of acidic and basic residues separated by a 4-nm stretch of neutral amino acids. The cap domain is extramembranous (27Go,28Go) and contains a vestibule with a maximum diameter of ~4.6 nm (23Go,29Go). The cap and stem are separated by a constriction with a diameter ~1.2 nm (23Go,30Go,31Go). Both openings of the channel are relatively large (diameter ~2.6 nm) (30Go).



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  		FIGURE 1  Molecular models of {alpha}HL. (A) Schematic illustration of sagittal section through the heptameric {alpha}HL 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 {alpha}HL 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 {alpha}HL channel (Protein Data Bank code 7AHL.pdb) were visualized with CS Chem3D Pro (CambridgeSoft). The final representations were produced with Adobe Photoshop.

 
By virtue of its relatively large internal lumen and the location of fixed charges inside the pore, the {alpha}HL channel is only weakly anion-selective and exhibits a mildly nonlinear and asymmetric current-voltage relationship (32Go–35Go). In contrast, narrow channels are likely to be dominated by electrostatic interactions between pore-permeant ions and the channel wall that results in a high degree of ion selectivity (36Go). Because the {alpha}HL channel has both wide and narrow regions inside its pore, it is an excellent candidate for ion selectivity studies.

The single {alpha}HL nanopore has also been used to study the transport of neutral (25Go,37Go–39Go) and charged polymers (5Go,11Go,40Go,41Go). Curiously, the charge state of ionizable amino acid side chains on the channel affects the dynamic partitioning of neutral polymers into the pore (42Go).

Our objective here was to determine the significance of the type and location of fixed charges on the selectivity, conductance (G), and G-V dependence of the {alpha}HL channel. The pioneering work by Akabas and colleagues demonstrated that cysteine scanning mutagenesis could be used to map the topology of an ion channel (43Go). Because the primary sequence of wild-type {alpha}HL has no cysteines, we produced many point cysteine mutants and determined their effects on the conducting properties of the {alpha}HL channel. The positions for some of these substitutions are shown in Fig. 1 B. We subsequently chemically modified these novel cysteine side chains with water-soluble sulfhydryl-specific reagents. The results suggest that the net charge of the channel wall is responsible for cation-anion selectivity of the {alpha}HL channel, whereas the balance of charges between the openings is the major determinant for the shape of the conductance-voltage curves.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 SUMMARY AND CONCLUSIONS
 ACKNOWLEDGEMENTS
 REFERENCES
 
1,2-Diphytanoyl-sn-glycero-3-phosphatidylcholine (DiPhyPC) was purchased from Avanti Polar Lipids (Alabaster, AL). Cholesterol, dithiothreitol (DTT), and 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) were purchased from Sigma Chemical (St. Louis, MO). 2-Sulfonatoethyl methanethiosulfonate (MTSES), 2-(trimethylammonium) ethyl methanethiosulfonate (MTSET), and 2-aminoethyl methanethiosulfonate (MTSEA), derivatives of methanethiosulfonate, were purchased from Toronto Research Chemicals (North York, Ontario, Canada). The other chemicals used in this study were analytical grade.

Electrophysiological measurements
Planar lipid bilayer membranes were formed as described elsewhere (39Go,44Go). Briefly, the two compartments of the Teflon cell were separated by a 25-µm-thick Teflon partition with an ~300-µm-diameter orifice that was pretreated with a 1% solution of hexadecane in n-pentane. DiPhyPC was dissolved at 5 mg/ml in pentane or hexane and spread at the air-water interface of the aqueous buffered solutions on both sides of the partition. Membranes were formed on the orifice by sequentially raising the level of the aqueous electrolyte solutions.

Channel-forming proteins were added to one side of the chamber (herein defined as cis). A negative applied electrostatic potential drives anions from the cis to the trans chamber. The membrane potential was maintained using Ag/AgCl electrodes in 3000 mM KCl, 3% agarose bridges. Milli-Q plus treated water (Millipore, Bedford, MA) with resistivity of 18 M{Omega} cm was used to prepare all buffer solutions. Unless stated otherwise, the standard solution contained 100 mM KCl, 1 mM DTT, and 30 mM Tris adjusted to pH 7.5 with HCl. The channel conductance, G, was measured in the presence of symmetric electrolyte solutions. To determine the G-V curve for single channels, voltage pulses that lasted for several seconds were applied.

The ion selectivities of the channels were estimated in the presence of a threefold electrolyte concentration gradient (e.g., with 100 mM KCl present on the trans side and 300 mM KCl on the cis side, both buffered to pH 7.5 with 30 mM Tris HCl), as described elsewhere (45Go). The transmembrane potential that is required to null the ionic current is defined as the reversal potential, Vrev. All potentials were corrected for electrode asymmetry (usually <0.3 mV) and for liquid junction potentials (calculated using the Henderson equation (46Go). A positive value for Vrev, with respect to the side containing the greater electrolyte concentration, indicates an anion-selective channel. To estimate the relative permeability ratios (PK/PCl), the Goldman-Hodgkin-Katz equation was employed. The PTRIS/PK was assumed to be equal to the ratio of their mobilities, uTRIS/uK ~ 0.4, (47Go).

The experiments were performed at room temperature (25 ± 2°C) under voltage-clamp conditions. The current was converted to voltage, digitized with a sampling frequency of 0.5 kHz, stored on a computer and analyzed off-line with the Whole Cell Electrophysiology Program (WCP V1.7b, J. Dempster, University of Strathclyde, Glasgow, UK).

Sulfhydryl reagents
The sulfhydryl reagents used in this study are small water-soluble sulfhydryl-specific reagents that have fixed positive or negative charges. They bind to cysteine side chains exposed to the aqueous phase. The reagents were usually applied to both compartments of the planar bilayer chamber at concentrations 0.5 mM, 2 mM, 1.0 mM, and 1.0 mM greater than the concentration of DTT in the solution for DTNB, MTSES, MTSET, and MTSEA, respectively. An increase in the reagent concentration over the values noted above caused no additional change in the values of the conducting properties of the channels reported below. The reaction of these reagents with a reduced cysteine side chain converts the –SH group to –SS-R, where R is the charged moiety, or The introduction of an additional charged group should affect the single-channel conductance if the chemically modified novel cysteine side chain either lines the channel lumen or is located adjacent to either channel entrance. A change in the channel conducting properties after reagent application served as a criterion for judging the accessibility of the point cysteine mutant side chain to the sulfhydryl reagent (43Go).

{alpha}HL proteins
Wild type {alpha}HL and single cysteine-substitution {alpha}HL mutants were prepared as previously described (48Go). The following mutant proteins were constructed and isolated: I7C, D108C, Y118C, F120C, N121C, G122C, V124C, G126C, D127C, D128C, T129C, G130C, K131C, I132C, G133C, G134C, L135C, I136C, G137C, A138C, N139C, V140C, S141C, and G143C. The proteins were stored at –70°C in the presence (mutants) or absence (wild-type {alpha}HL) of 5 mM DTT. Before the electrophysiological experiments, the mutant versions of {alpha}HL were reduced by incubation with 30 mM DTT for 20 min. Stock solutions of the reduced proteins were then stored at 4°C for a maximum of 5 days, and diluted 10- to 50-fold before the experiments.

The positions of each amino acid side chain along the channel axis, with respect to the trans entrance, were estimated from the {alpha}HL channel crystallographic structure (7AHL.pdb) using Swiss-Pdb Viewer version 3.7 (49Go) and CS Chem3D Pro (CambridgeSoft, Cambridge, MA).


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
SUMMARY AND CONCLUSIONS
 ACKNOWLEDGEMENTS
 REFERENCES
 
Cysteine substitution and G-V dependencies of channels
Analytical studies (19Go,50Go,51Go) demonstrated that fixed charges on membrane surfaces (in the absence of channels) or inside channels could cause rectification of ionic current. Subsequent experiments on a K+ channel (52Go) and the {alpha}HL channel (28Go,32Go,34Go,53Go) showed that the charge on the membrane surface could alter ion selectivity and the G-V relationship. Therefore, to exclude the effects of fixed charges on the membrane, we used zwitterionic phosphatidylcholine bilayers. The trans channel opening adjacent to one of the membrane surfaces (8Go,28Go) was used as a zero-position reference point.

The conductance, G-V lineshape, ion selectivity, and other {alpha}HL channel properties are sensitive to the pH and bathing solution electrolyte concentration (2Go,4Go,28Go,32Go,34Go,35Go,53Go–55Go). Fig. 2 demonstrates that increasing the 1:1 electrolyte concentration decreases the asymmetry (with respect to the sign of the applied potential) of the G-V curves for wild-type {alpha}HL channels. Interestingly, current rectification was observed even at 4000 mM KCl. These results suggest that the charges lining the pore walls mainly control the ability of mobile ions to be transported through the {alpha}HL channel (19Go). It follows that changes in the sign, magnitude, and/or position of charges lining the channel lumen result in changes to the local electrostatic environment and the channel conducting properties. To better reveal the role of these charges, in the majority of experiments, we used aqueous solutions with a relatively low ionic strength (i.e., 100 mM KCl) because the effective Debye length is large (~1 nm). The experiments are more difficult to carry out in solutions with even lower ionic strengths because the {alpha}HL channel tends to gate quickly (J. J.Kasianowicz, unpublished observation).



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  		FIGURE 2  Voltage dependence of wild-type {alpha}HL 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.

 
All of the {alpha}HL mutants reported here were divided into three groups. The first group included the mutants (I7C and D108C) with substitutions in the cap domain. Mutants in two other groups contained a novel cysteine at even-numbered positions or odd positions of the ß-barrel stem region, respectively. In all cases, the addition of wild-type {alpha}HL or any of the mutants at concentrations of ~1–10 ng/ml to the cis aqueous compartment led to a stepwise increase in the ionic current (Fig. 3). Each step corresponds to the formation of a single channel.



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  		FIGURE 3  Conductance histogram of single channels formed by the genetically engineered protein {alpha}HL G134C. The inset illustrates a typical current recording of single channels spontaneously formed by G134C {alpha}HL 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.

 
{alpha}HL reconstitutes into the membrane in the same orientation each time, most likely because the large cap domain cannot traverse the membrane. The well-defined asymmetry in the {alpha}HL channel G-V curve (25Go,28Go,32Go,33Go) serves as a test of this ansatz. The {alpha}HL channel conductance, as determined from the step increases in current, varies slightly from channel to channel (see, e.g., Krasilnikov and colleagues (25Go,30Go,32Go,41Go)). Therefore, we recorded the formation of several hundred individual (i.e., distinct) ion channel formation events. The step conductances were analyzed and used to build cumulative probability density amplitudes. Examples of an ionic current recording and the step current probability density amplitudes are shown in Fig. 3. The histograms usually have a well-defined peak that we fitted with a Gaussian curve. The value of the most probable channel conductance obtained for each mutant {alpha}HL is defined herein as a standard. Only the channels with a conductance close to this nominal value were used to study G-V dependencies in the single-channel experiments (i.e., we ignored the much less probable lower conductance states). When membranes containing many channels were used, the data were normalized to the number of channels present in the membrane. As was noted above, the {alpha}HL channel can gate from the fully open state to closed states (2Go,54Go,56Go–58Go). If a channel gating event occurred during the course of an experiment, we did not analyze subsequent step increases in current. Thus, we only present data that correspond to fully open channels.

Because Ohm's law is a reasonable first approximation for the ionic conductance of large channels, we expected the channel properties to depend on the type of {alpha}HL mutant. For example, a novel cysteine side chain could alter the channel conductance simply because its volume is different from the side chain it replaced. In addition, changes in the fixed charge distribution inside or near the pore would change the transmembrane electrostatic potential profile and, hence, the barriers and wells for pore-permeant mobile ions. Examples of G-V curves for the point cysteine mutants and wild-type {alpha}HL are presented in Fig. 4. Of the 24 mutants examined, only six mutations that are close to the trans channel entrance (i.e., amino acid side chains at positions 126, 128, 129, 130, 131, and 133 (Fig. 1 A)) have considerable influence on the channel conductance. Except at position 130 (see the experimental study of Krasilnikov et al. (53Go)) and positions 128 and 131 (see the theoretical study of Noskov et al. (19Go)), none of the other three mutations were reported to have a major effect on {alpha}HL channel properties without subsequent chemical modification. In addition, only the channel formed by D128C demonstrates an inversion in the conductance-voltage dependence. The latter result is consistent with the assumption (19Go) that D128, although presumably in a hydrophobic zone (see below), is in a charged form and contributes to the channel conductance rectification.



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  		FIGURE 4  Steady-state conductance-voltage relationships of single channels formed by wild-type and novel point cysteine mutant versions of {alpha}HL. 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.

 
The G-V curves of the channels formed by other mutants were only slightly different from the control data for the wild-type {alpha}HL channel. Thus, novel cysteines at those positions did not significantly affect channel structure. To better visualize the effects of the amino acid substitutions, the asymmetry of the G-V curves at ±100 mV is shown in Fig. 5.



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  		FIGURE 5  Effect of single cysteine substitutions on the {alpha}HL 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 {alpha}HL 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 {alpha}HL 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.

 
The change in asymmetry (or rectification ratio, G–100 mV/G+100 mV) of the ion channel conductance is greater when the cysteine substitution is made closer to the trans pore entrance. This is true for the substitutions at both odd- and even-numbered positions in the ß-barrel. The results for odd-numbered positions are sensible because the crystal structure suggests that the side chains of these amino acid residues face the pore lumen (23Go). The effectiveness of some cysteine substitutions at even-numbered positions is also plausible because they are close to the trans pore entrance and apparently not buried in the membrane.

In general, the point cysteine substitutions, except for D127C and D128C, increase the asymmetry of the G/V curves. An increase in the rectification ratio was observed for channels formed by mutants in which a positively charged (K131) or neutral residue (especially if it is close to the trans opening) was substituted with Cys having a weak negative charge. These results suggest that the electric charge magnitude and location affect the value of the ionic current rectification. The pKa of the cysteine sulfhydryl group is generally alkaline (59Go). Thus, at the pH value in the experiments reported here, the substitution of any neutral amino acid residue with cysteine would introduce a negative charge at the position. This novel charge should be less than that of the carboxyl group of Asp, because the pKa of Asp in the bulk is ~4 (i.e., several pH units lower than the pH of the solutions used in this study). Indeed, the G-V curve of the channel formed by D128C is significantly changed. Thus, we suggest that a negative charge close to the trans pore entrance increases the asymmetry in the G-V curve, whereas one close to the cis opening decreases it. A cysteine substitution at position 7 should therefore decrease the asymmetry of the G-V curve. However, this was not observed for channels formed by I7C; the weak negative charge of Cys was not strong enough to evoke a change because the diameter of the {alpha}HL channel at this location is considerably larger than that in the stem region. However, a distinct change was observed when a greater negative charge was introduced at this position by targeted chemical modification (24Go). The results suggest that the asymmetry (and the G-V curve) is mainly determined by the asymmetric distribution of charges in the two halves of the channel. Negative charge predominates in the trans opening, and positive charge predominates in the cis entrance. These findings are consistent with a recent theoretical study (19Go).

Cysteine substitution and cation-anion selectivity of the channel
The results of cation-anion selectivity measurements for the channels formed by wild-type {alpha}HL and the mutant versions are summarized in Fig. 6. Cysteine substitution changed the channel selectivity, and the degree of the change depended on the location of the novel Cys. Point cysteine substitutions at odd-numbered positions caused a larger change in selectivity. However, mutations at several even-numbered positions also elicited changes in ion selectivity. These effects were greatest when the substitution was made close to the trans opening. Cysteine substitutions for negatively charged carboxyl groups of Asp-127 and Asp-128 increased the anion selectivity of the channel. All other substitutions led to a decrease in the anion selectivity or even reversed it (i.e., K131C, G133C, and G126C). The strong cation selectivity of the channel formed by K131C, the mutant with the most prominent change, is easy to explain. It results from the substitution of the positive charge of the Lys side chain with the weak negative charge of Cys. The effects due to G133C and G126C were moderate. Nevertheless, the introduction of even a weak negative charge at these positions impacts the channel ion selectivity. We cannot completely rule out the possibility that these substitutions also result in a local reorganization in stem structure. Cysteine substitutions in the cap region (D108C and I7C) made anion selectivity of the channel weaker than the channel formed with wild-type {alpha}HL. The asymmetry of the D108C channel mutant is only slightly larger than that of the wild-type {alpha}HL channel, whereas the asymmetry of the I7C channel was practically unaltered.



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  		FIGURE 6  Effect of single cysteine substitutions on the reversal potential of {alpha}HL 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 {alpha}HL. 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 Vrev obtained in 5–7 separate experiments.

 
Based on the results above, we suggest that the net integrated charge is responsible for cation-anion selectivity of the {alpha}HL channel, whereas the balance of charges between the openings is crucial for determining the asymmetry of the G-V curves. To test this hypothesis, we used reagents that interact specifically with the sulfhydryl group of cysteine and that add either a full positive or negative charge at each cysteine side chain.

Cysteine derivatization and G-V dependencies
The conversion of the weak negative charge on cysteine to either a more negative or positive value should produce significant changes in the local electrostatic potential profile. In an attempt to affect such changes, each of the point cysteine mutants was subjected to targeted chemical modification with specific sulfhydryl reagents. These experiments were conducted with preformed heptameric channels and channels formed by chemically modified monomers of {alpha}HL. The reagents (when applied on the preinserted channels) rapidly affected the channel conducting properties, and new steady-state values were reached within minutes. Changes in the channel conductance and rectification after reagent application served as a criterion for judging the accessibility of the sulfhydryl group. The effect was evident in bilayers that contained either multiple or single channels. In the case of multiple channels, the reagent-induced change to the membrane current was smooth (data not shown) and reflected the aggregate change in the conductance of individual ion channels. Similar to our findings in an earlier study (24Go), the reagents caused abrupt, cascade-like changes in the channel conductance (Fig. 7 A), suggesting the rapid, consecutive modification of the cysteines within the individual heptameric pores. The time-resolved cascade of changes in conductance could be observed in the presence of relatively small reagent concentration (24Go). Here, our goal was to rapidly derivatize the available sites (Fig. 7 B). It needs to note that the rate of the cysteine derivatization depends on the type of reagent also. The rate was higher with MTS reagents than with DTNB. When the steps were resolved, the time interval between them varied from channel to channel, reflecting the stochastic nature of the process. As was shown elsewhere (24Go) the maximum number of the stepwise changes in the {alpha}HL channel conductance was seven. In general, the number of steps we observed was <7. When channels reached the new level of conductance in <7 steps, some of the steps were approximately twice (or more) the magnitude of single steps (Fig. 7 A). We assume these events correspond to the simultaneous modification of two or more sulhydryl groups. The seven stepwise changes in channel conductance confirm that {alpha}-toxin channel is a heptamer and suggests that all cysteines in the channel are derivatized by the reagents. The conclusion was confirmed with experiments in which mutated {alpha}HL monomers were first exposed to an excess of one of the sulfhydryl reagents and then allowed to form channels. For all mutants with a cysteine at an odd-numbered position in the stem region, both chemical modification methods gave virtually identical results. We therefore conclude that all seven novel cysteines for each of these mutants are accessible to, and modified by, the reagents. The effects of sulfhydryl reagents could always be reversed by DTT, and the channels then exhibited properties identical to those of the unmodified mutated toxins. This result confirms that the changes are the result of the reversible modification of the sulfhydryl groups and not due to an irreversible conformational change of the channel.



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  		FIGURE 7  Single-channel recordings of G143C-{alpha}HL 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.

 
The shape and the rectification of the preinserted, assembled oligomeric channel formed by mutated {alpha}HL treated with MTSES and MTSET at low ionic strength (100 mM KCl) are shown in Figs. 8 and 9. The effects of DTNB and MTSEA were analogous to those of MTSES and MTSET, respectively (data not shown). Specifically, the addition of full negative charges (sulfate or carboxyl groups, MTSES or DTNB, respectively) in the stem region made the G-V curves more asymmetric, and full positive charges (trimethylammonium or amino groups, MTSET or MTSEA, respectively) decreased the rectification. Also, the influence of these novel full charges depends on the position of the charged group. The effect is not particularly strong for charges added at the trans entrance. However, the effect first increases as the charges are located into the pore (reaching its maximum value at ~1 nm from the trans opening) and then decreases at distances >3 nm.



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  		FIGURE 8  Conductance-voltage relationships of channels formed by the odd-numbered single cysteine {alpha}HL 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%.

 


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  		FIGURE 9  Effect of sulfhydryl reagents on the {alpha}HL 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 {alpha}HL channel. The sequence of mutants from left to right is T129C, G130C, K131C, D127C, G133C, L135C, G137C, N121C, N139C, S141C, and G143C.

 
In contrast, the results obtained with the even numbered mutants were more complex. Except for G130C, sulfhydryl groups at these positions were not always accessible in preformed channels. However, chemical modification of monomeric {alpha}HL mutants before channel formation demonstrated that some even-numbered positions (i.e., 124, 126, 128, 132, 134, 136, 138, and 108) are accessible to the reagents because such derivatized mutants form channels with properties considerably different from those of channels formed by original monomeric {alpha}HL mutants (data not shown). Apparently, at those particular positions, the charged moieties introduced by the sulfhydryl (SH)-specific reagents cannot be accommodated. The subsequent addition of DTT to these channels did not reverse the effects of derivatization by sulfhydryl agents.

Some of the other even-numbered {alpha}HL mutants (i.e., Y118C, F120C, G122C, and V140C) are relatively insensitive to the reagents, even in monomeric form. In contrast, the sulfhydryl group of Cys-130 was accessible in monomeric {alpha}HL (aqueous solution) and in preformed channels.

The I7C channel was also sensitive to the reagents. In contrast to the stem region, the introduction of a strong negative charge by DTNB and/or MTSES in the cap domain made the G-V curves less asymmetric. The corresponding rectification ratios were 1.55 ± 0.06 and 1.1 ± 0.02, respectively. An attempt to introduce a positive charge at this position of the preformed channel with MTSEA and MTSET was apparently unsuccessful. In that case, we did not observe a significant change in the rectification. The positive charge on Lys-8 may electrostatically repel the positively charged reagent. The effects of the negatively charged sulfhydryl reagents on the I7C mutants were reversed by DTT.

Cysteine derivatization and cation-anion selectivity
The effects of charge introduction on cation-anion selectivity of the {alpha}HL channel are summarized in Fig. 10. In this set of the experiments, the channels were first formed by mutant {alpha}HL in lipid bilayer membranes, and then water-soluble sulfhydryl-specific reagents were applied. The cysteines at even-numbered positions were virtually inaccessible to SH reagents. A weak change in the channel selectivity was observed only in the case of Cys-130, which is located close to the trans entrance.



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  		FIGURE 10  Effects of sulfhydryl reagents on the reversal potential of channels formed by single cysteine {alpha}HL 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 PK/PCl 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 {alpha}HL. Each point represents the mean value (± SD) of Vrev obtained in 5–7 independent experiments.

 
As expected, selectivity changes were observed for all odd-numbered sites in the stem region that we tested. The data provide additional support for the ß-barrel structure of the pore-forming stem region (23Go,60Go). The extent of the selectivity changes caused by the chemical derivatization was virtually identical to that obtained with channels formed by the same odd-numbered mutants derivatized in monomeric form in aqueous solution. The effectiveness of the novel charges was dependent on their location along the channel axis. Relatively weak effects were found for charges located close to the channel opening. The effect of charges increased with distance, and at a distance of 1–1.5 nm from the trans opening (i.e., the end opposite the large cap domain of {alpha}HL) Vrev was maximal at ~+21 mV (MTSET) or at –24 mV (MTSES), with relative permeability ratios, PK/PCl, of ~0.001 and ~170, respectively, whereas wild {alpha}HL channel is weakly anion-selective with Vrev ~ 7.6 mV and PK/PCl ~ 0.46. The maximal values of Vrev for the derivatized {alpha}HL channels are close to the Nernst potentials (~+22 mV and ~–25 mV for Cl– and K+, respectively). Thus, the derivatized {alpha}HL channels are highly selective for anions or cations, depending on the sign of the novel charge. Charges located further in the pore interior have a greater influence on selectivity.

The position of the novel charge affects Vrev and the asymmetry of G-V curves differently. After an initial increase, Vrev remains large and nearly constant (Fig. 10 A). In contrast, the change in the asymmetry vanishes quickly with distance from the trans pore entrance (Fig. 9). The results suggest that the net (integrated) charge is responsible for cation-anion selectivity of the {alpha}HL channel, whereas the balance of charges between the openings is crucial for determining the G-V curves.

The introduction of a strong negative charge (i.e., with DTNB or MTSES reagents) into the cap domain (I7C channel) changes the channel selectivity in a manner consistent with modification of the stem region (the channel became more cation-selective). The change in Vrev (from 7.6 ± 0.7 mV to 5.8 ± 0.7 mV) was moderate, but statistically reliable (P < 0.05). The large pore radius in the cap region may be responsible for the less striking result. As mentioned above, the positively charged reagents (i.e., MTSET and MTSEA) caused no discernable effects on I7C channel properties.

Stem radius and the charge effectiveness
The long-range action of a charge depends on the ionic strength of the supporting electrolyte and may be described approximately with the Debye equation for planar surfaces. Thus, the placement of charges close to the pore openings will be readily affected by changes in the bulk ionic strength. How far into the pore would changes in the bulk aqueous solution be felt? To address this question, the reversal potential was also measured in the presence of a 3000 mM/1000 mM (cis/trans) KCl gradient (Fig. 10 B). In contrast to the results obtained at lower KCl concentration (Fig. 10 A), the Vrev-distance dependencies have a minimum at ~2.5 nm from the trans opening. This behavior was considerably different from that observed at low salt concentrations for the point cysteine mutants (Fig. 6). We draw two conclusions: the increase in KCl concentration in bathing solution decreases the long-range action of the charges even though they are far from the opening, and that additional factor(s) contributed to the value of the reversal potential, Vrev. As shown above, a ring of charge introduced into the wider cap vestibule caused only slight changes to the {alpha}HL channel conducting properties. Are the charges added to the stem region located at a different distance from the channel axis? The stem of {alpha}HL channel is a right hand ß-barrel. The radius, measured from {alpha}-carbon positions, is assumed to be ~1.3 nm. However, the detailed analysis of the channel radii at each of the odd-numbered side-chain {alpha}-carbon atoms, performed with the Swiss-Pdb Viewer version 3.7 (49Go) and CS Chem3D Pro, demonstrates that they are not constant. The variation of the radius with distance from the trans pore entrance is nearly a mirror image of the distance dependence of Vrev (Fig. 11 A). Thus, there is an inverse correlation between these two parameters (Fig. 11 B), which suggests that the geometry determines the effectiveness of the charges even in such confined spaces as water-filled pores of nanometer dimensions.



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  		FIGURE 11  Interrelation between the variation of the reversal potential of ion channels formed by cysteine-substituted derivatized {alpha}HL mutants and the radius of the wild-type channel. (A) The variation of the reversal potential ({circ}, {triangleup}) of ion channels built by cysteine-substituted derivatized {alpha}HL mutants and the radius ({blacksquare}) of the channel with the distance from the trans opening . {triangleup}, MTSES treatment; {circ}, MTSET treatment. The data are the absolute value of the difference in Vrev 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 {alpha}-carbon of each odd amino acid residue in the stem region using the {alpha}HL channel crystallographic structure (7AHL.pdb), the Swiss-Pdb Viewer version 3.7 (49Go), 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.

 

   SUMMARY AND CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 SUMMARY AND CONCLUSIONS
ACKNOWLEDGEMENTS
 REFERENCES
 
We used cysteine-scanning mutagenesis to better understand the basis of ion selectivity and ionic current rectification in a channel with a known three-dimensional structure. The aim of this study was to determine experimentally the significance of both the charge type and its position within the ion conduction pathway on the selectivity, conductance, and G-V lineshape of the {alpha}HL channel. The choice of {alpha}HL channel was also motivated by its potential importance for biotechnology applications.

Several attempts were earlier made to describe theoretically these {alpha}HL channel properties, where qualitative (28Go,35Go) or nearly quantitative (19Go) descriptions were achieved assuming that the fixed charges inside the pore and near the pore's entrances affect the channel properties. We have determined the G-V relationship and cation-anion selectivity of channels using 24 cysteine-substituted {alpha}HL mutants with or without derivatization of Cys with sulfhydryl-specific water-soluble reagents. The conditions we used (low ionic strength and net neutral planar lipid membranes) were optimal for revealing even the influence of weak charges. In this study, we demonstrate how the magnitude of the effect of charges depends on the position of the charges along the longitudinal channel axis.

The selectivity and the asymmetry of the conductance-voltage relationship of {alpha}HL channel depends on the bulk aqueous solution ionic strength, which suggests that the charges facing the aqueous phase inside the nanoscopic pore play a key role in determining these channel properties. Among the 24 point cysteine mutants reported here, only six mutations that are close to the trans opening (i.e., positions 126, 128, 129, 130, 131, and 133 in the stem region) have a significant influence on the channel conductance. Except at position 130 and positions 128 and 131, none of the other three mutations were reported previously to have a major effect on {alpha}HL channel properties without chemical modification.

Sulfhydryl-specific water-soluble reagents changed the properties of preinserted, assembled oligomeric channels formed by odd-numbered mutants with Cys in the stem region. Among the channels formed by even-numbered mutants, the G130C channel was unique in having properties that were altered by the reagents. The changes in properties of preinserted channels are most likely the result of the addition of novel fixed charges to the pore wall (the effects of sulfhydryl reagents on these mutants could always be reversed by DTT).

The treatment of monomeric {alpha}HL mutants with specific sulfhydryl reagents in solution before channel formation demonstrated that some even-numbered positions (124, 126, 128, 132, 134, 136, 138, and 108) form channels with properties considerably different from those of channels formed by original monomeric {alpha}HL mutants. In the case of these derivatized even-numbered mutants, the effects of sulfhydryl reagents could not be reversed by treatment of the channels with DTT. Some of the other even-numbered {alpha}HL mutants (Y118C, F120C, G122C, and V140C) appear to be insensitive to the reagents.

The effectiveness of the novel charges depends on both their type and location along the channel axis. The introduction of a strong negative charge in the trans part of the pore (stem region) made the channel more selective to cations and increased the asymmetry of the G-V curve. In contrast, the introduction of a positive charge had the opposite effect. Relatively weak effects were observed for mutants with charges located close to the channel openings. The effect of charges initially increased with distance from the trans opening and had a maximum influence on the asymmetry of G-V curves or saturated close to the Nernst potentials (Vrev) at a distance of 0.8–1.5 nm from the trans opening. Thus, it is possible to create an ion-selective filter for relatively large water-filled pores of nanometer dimensions by the introduction of a ring of charge. The charges have to be located in regions with smaller radii inside the pore to have a stronger influence on the {alpha}HL channel selectivity and to be located close to pore openings to affect the channel G-V dependence. These charged rings not only affect G-V dependence and the channel's selectivity to small mobile charges, they also have a strong influence on polynucleotide transport through the {alpha}HL channel (S. E. Henrickson, O. V. Krasilnikov, A. Valeva, and J. J. Kasianowicz, unpublished observations).

In contrast to the results obtained with charges added to the stem region, the introduction of a negative charge in the cap domain made the G-V curves less asymmetric and the channel more cation-selective. The results suggest that the net (integrated) charge is responsible for cation-anion selectivity of the {alpha}HL channel, whereas the balance of charges between the openings is crucial for determining the G-V curves.

An increase in the KCl concentration in the bulk determines the mobile charge concentration within the channel. The mobile charges screen the fixed charges added to the pore wall, even when they are relatively far from the pore entrances.

The stem region of {alpha}HL channel is a right-hand ß-barrel. However, the radius, measured from {alpha}-carbon positions of each odd amino acid residue in the stem, is not constant. The value of the radius deduced from the crystal structure correlates inversely with the effectiveness of the novel charges on ion selectivity. Thus, the local properties of the channel influence the effectiveness of the charges in an ion channel with nanometer-scale dimensions. We expect that the results obtained here will be applicable to other biological or synthetic water-filled nanopores.


   ACKNOWLEDGEMENTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 SUMMARY AND CONCLUSIONS
 ACKNOWLEDGEMENTS
REFERENCES
 
We are grateful to Dr. Elbert Lee (National Institute for Physiological Sciences, Okazaki, Japan) for helpful suggestions on the manuscript. Inclusion of suppliers and manufacturers of materials and equipment is for completeness and does not constitute endorsement by the National Institute of Standards and Technology. This work is dedicated to the memory of Professor Gianfranco Menestrina, one of the pioneers in the study of pore-forming toxins.

This study was partially supported by Conselho National de Desenvolvimento Cientifico e Tecnologico (Brazil). This work was sponsored in part by the National Institute of Standards and Technology Advanced Technology Program, and the National Institute of Standards and Technology "Single Molecule Manipulation and Measurement" program, and the National Science Foundation (Nanoscale Interdisciplinary Research Team grant CTS-0304062).

Submitted on May 12, 2005; accepted for publication July 26, 2005.


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 SUMMARY AND CONCLUSIONS
 ACKNOWLEDGEMENTS
 REFERENCES
 
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