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Langmuir 1996, 12, 2058-2067
Influence of Biologically Active Compounds on the Monomolecular Gramicidin Channel Function in Phospholipid Monolayers Andrew Nelson Plymouth Marine Laboratory, Citadel Hill, Plymouth, PL1 2PB, U.K. Received September 20, 1995. In Final Form: January 2, 1996X The effect of biologically active compounds on ion channel transport within a self-assembled lipid monolayer system was investigated by examining interactions between some bioactive compounds and the gramicidinmodified lipid monolayer as a membrane model in solutions of different cation concentration and composition. Techniques used were ac voltammetry to measure the capacitance of the layers and cyclic voltammetry (CV) and chronoamperometry to study Tl electrochemistry. The CV results tend to support the proposed model that nonlinear diffusion effects are not significant in the electrochemistry of Tl within the time scale of the experiment. As a result the electrode process is treated as a CE mechanism where the rate limiting step is the translocation of the ion in the channel. Complications arise because there is competition between the electroactive ion, Tl+, and the univalent electrolyte ion to enter the channel under the influence of the applied potential field. Values of the permeability rate constants have been derived from the electrochemical data. Results indicate that many hydrophobic additives in the lipid layer in particular polyaromatic and polyconjugated compounds selectively give rise to an alteration in the permeability of gramicidin-modified DOPC monolayers to Tl+. Up to 5-fold increase in gramicidin-mediated permeability is noted in monolayers with added retinol, which is similar to that seen in the presence of a similar concentration of negatively charged lipids (PS) in the layer. The chlorine-substituted aromatic pesticide DDT causes an apparent depression in the permeability. It is proposed that the variations of permeability to Tl+ due to additives in the layer are predominantly caused by the effect of the compounds within the monolayer on the energy barrier to the translocation of the ion within the channel. This occurs because the compounds alter the image forces acting on the ion passing through the channel as well as directly interacting with the ion itself.
Introduction It has always been a controversy whether hydrophobic bioactive compounds, e.g., anesthetics, manifest their activity by interacting directly with specific hydrophobic receptors1-3 or more generally with the lipid component of the biological membrane.4,5 Some experiments have been performed which have shown anesthetics to interact with a specific protein receptor molecule.3 It has also been found that anesthetics and related hydrophobic compounds interact with both the lipid component of natural biological membranes5,6 and lipid bilayers influencing channel function therein.6,7 Generally a decrease in the gramicidin channel conductivity has corresponded to an accumulation of the toxic compound in the bilayer. An increase in the thickness of the bilayer contributing to a decreased stability of the bimolecular gramicidin channel has been proposed to account for this effect.7,8 This explanation has been used to clarify the suppressive effect from hydrophobic toxic compounds on ion channel function in natural biological membranes.4 With respect to the stability of gramicidin, more recent studies have investigated the forms of gramicidin in lipid vesicles and have identified two types of gramicidin: a nonconductive double-stranded dimer and a conductive single-stranded monomer.9,10 The relative proportion of the two forms in membraneous structures depends on X
Abstract published in Advance ACS Abstracts, March 15, 1996.
(1) Franks, N. P.; Lieb, W. R. Nature 1982, 300, 487-493. (2) Franks, N. P.; Lieb, W. R. Nature 1988, 333, 662-664. (3) Franks, N. P.; Lieb, W. R. Nature 1986, 319, 77-78. (4) Hendry, B. M.; Elliott, J. R.; Haydon, D. A. Proc. R. Soc. London 1985, B224, 389-397. (5) Elliott, J. R.; Haydon, D. A. Nature 1986, 319, 77-78. (6) Haydon, D. A.; Hendry, B. M.; Lewinson, S. R.; Requena, J. Nature 1977, 268, 356-358. (7) Haydon, D. A. Ann. N.Y. Acad. Sci. 1975, 284, 2-16. (8) Hladky, S. B.; Haydon, D. A. Curr. Top. Membr. Transp. 1984, 21, 327-372.
0743-7463/96/2412-2058$12.00/0
many factors but in particular on the solvent history of the gramicidin and on the lipid composition.9-12 In addition peptide-peptide intramolecular and lipid-peptide intermolecular interactions regulate the gramicidin channel structure.13 It has been shown very recently that the tryptophan residues of the gramicidin helix are critical in orienting the peptide with respect to a lipid bilayer surface; in addition they are implicated for a major role in inserting the peptide into the bilayer.14 Clearly the aromatic ring of tryptophan and the ability of the neighboring polar indole group to take part in hydrogen bonding play an important role in this. As well as these rather specific studies on gramicidin channel function, a comprehensive series of theoretical studies have been carried out on model ion channel function including studies of the dependence of ion transport on the structure15,16 and electrostatic17,18 properties of the channel, the lipid environment,15-17 and the electrolyte composition.18,19 This present investigation has been undertaken therefore to reassess the link between the interaction of hydrophobic bioactive compounds with the lipid backbone of membranes6 and the gramicidin channel function in lipid membranes using an experimental approach. The aim (9) Killian, J. A.; Urry, D. W. Biochemistry 1988, 27, 7295-7301. (10) Cox, K. J.; Ho, C.; Lombardi, J. V.; Stubbs, C. D. Biochemistry 1992, 31, 1112-1118. (11) Salom, D.; Abad, C.; Braco, L. Biochemistry 1992, 31, 80728079. (12) Killian, J. A.; Prasad, K. U.; Hains, D.; Urry, D. W. Biochemistry 1988, 27, 4848-4855. (13) Scarlata, S. F. Biochemistry 1991, 30, 9853-9859. (14) Ketchem, R. R.; Hu, W.; Cross, T. A. Science 1993, 261, 14571460. (15) Jordan, P. C. Biophys. J. 1984, 45, 1091-1100. (16) Jordan, P. C. Biophys. J. 1984, 45, 1101-1107. (17) Jordan, P. C. Biophys. J. 1983, 41, 189-195. (18) Cai, M.; Jordan, P. C. Biophys. J. 1990, 57, 883-891. (19) Jordan, P. C.; Bacquet, R. J.; McCammon, J. A.; Phouc, T. Biophys. J. 1989, 55, 1041-1052.
© 1996 American Chemical Society
Ion Channel Transport
was to further the understanding into how and by what mechanism a varying lipid environment influenced the gramicidin channel activity in simple electrochemical membrane models. In this study the gramicidin-modified lipid monolayer adsorbed on a mercury electrode within an electrochemical cell was used.20 This membrane model which is a development of that of Miller21 has some advantage over free standing bilayer lipid membranes in having an inherent mechanical stability. In addition using conventional electrochemical methods, the electric potential can be controlled and measured precisely. The lipid monolayers can be rapidly renewed on fresh mercury electrode surfaces and the reproducibility of successive structures on the electrode is good. Gramicidin forms monomolecular channels in lipid monolayers which conduct univalent ions.20 This system has already been used to study fixed charge and solution effects on ion permeability.22 Previous studies have also looked at the interaction of hydrophobic organic compounds with the pure lipid monolayer.23 The present work combines these two approaches in order to investigate the influence of bioactive hydrophobic organic compounds within the phospholipid monolayer system of dioleoylphosphatidylcholine (DOPC) on the monomolecular gramicidin channel function. Representatives from the following groups of compounds were studied: (a) polycyclic aromatic hydrocarbons (PAH), (b) polyconjugated compounds, (c) neurotoxic pesticides, and (d) cholesterol. The effect of phospholipid mixtures of DOPC and the negatively charged lipid phosphatidylserine (PS) on gramicidin channel activity was also investigated. In addition, experiments were undertaken not only with monolayers of different composition but also with solutions of varying cation concentration and composition. The following approach was taken in this study. Differential capacity-potential plots of the coated electrode were measured to assess the structure of the adsorbed layer. Cyclic voltammetry (CV) of the Tl(I)/ Tl(Hg) couple at the coated electrodes was carried out to investigate the nature of the electrode mechanism and to obtain kinetic data. Potential step experiments were done to substantiate the conclusions from the CV experiments. Experimental Section Electrolytes were fully deaerated with special grade argon before each experiment and a blanket of argon gas was kept above the electrolyte during the experiment. Monolayers of DOPC and mixed monolayers of DOPC and additive were prepared as before by spreading a pentane solution of DOPC and by mixing DOPC and the required mole fraction of additive compound in pentane and spreading this solution respectively at the gas-water interface in the electrochemical cell.23 Such monolayers are referred to in the text as the lipid-mole fraction additive. All additives were initially prepared as acetone working solutions. Monolayers at the gas-water interface were modified with gramicidin (Sigma) by adding 0.128 µmol dm-3 gramicidin to the electrolyte from a methanol stock solution in all experiments. The electrolyte was subsequently stirred for 5 min to allow full equilibration of the gramicidin with the monolayer. A fresh mercury drop electrode of area 0.0088 cm-2 was coated with the monolayer from the gas-water interface for each experiment. All potentials in this paper are reported versus Ag/AgCl 3.5 mol dm-3 KCl. Differential capacity-potential plots were obtained through the use of out-of-phase (90°) ac voltammetry carried out with a (20) Nelson, A. J. Electroanal. Chem. 1991, 303, 221-236. (21) Miller, I. R. In Topics in Bioelectrochemistry and Bioenergetics; Milazzo, G., Ed.; John Wiley and Sons: Chichester, 1981; Vol. 4, pp 161-224. (22) Nelson, A. J. Chem. Soc., Faraday Trans. 1993, 89, 2799-2805. (23) Nelson, A.; Auffret, N.; Borlakoglu, J. Biochim. Biophys. Acta 1990, 1021, 205-216.
Langmuir, Vol. 12, No. 8, 1996 2059
Figure 1. Structures of molecules added to the lipid layer in this study: 1, benzo[a]pyrene (B-a-p); 2, (a) All-trans- and (b) 13-cis-retinol; 3, 1,1-bis(4-chlorobiphenyl)-2,2,2-trichlorethane (DDT); 4, cholesterol. Metrohm 506 Polaricord. The settings for this were frequency 75 Hz, peak amplitude 0.0085V, and scan rate 0.006 V s-1, and the current was calibrated as described previously.24 The equipment and methods used for performing cyclic voltammetry (CV) were precisely as previously reported.20,22 A CV of 4 × 10-4 mol dm-3 Tl(I) in solution at the monolayer-coated electrode prior to penetration by gramicidin was carried out as a control. CV of 4 × 10-4 mol dm-3 Tl(I) in solution at a range of scan rates from 0.2 to 20 V s-1 and CV of 4 × 10-4 mol dm-3 Cd(II) in solution at scan rate 0.2 V s-1 in a few experiments were carried out at the gramicidin-modified coated electrode in electrolytes: 0.1 mol dm-3 KCl and 1 mol dm-3 KNO3 at pH 7.2 buffered with 0.01 mol dm-3 phosphate and 0.05 mol dm-3 and 0.5 mol dm-3 Mg(NO3)2 at pH 5.8 buffered with 0.01 mol dm-3 acetate. Experiments in Mg2+ electrolyte were carried out in acetate buffer to avoid reaction of Mg2+ with phosphate. Voltammetric experiments were carried out in a few cases with 8 × 10-4 mol dm-3 Tl(I) in solution and also 0.021 and 0.01 µmol dm-3 gramicidin in the electrolyte from a methanol stock solution. Chronoamperometric experiments were done at the same coated electrodes and in the same solutions as the CV experiments by applying a step potential for 25 ms from -0.2 V to -0.6 and -0.7 V using a Hewlet-Packard function generator attached to the E506 Polarecord and recording the resulting current-time trace on a Hameg storage oscilloscope. The current-time traces from the chronoamperometry experiments were semi-integrated using the method of Oldham.25 Although a spherical electrode was used in this work, the effects of sphericity were ignored in the subsequent analysis. The reason for this was that all electrochemical measurements were carried out at sufficiently short time scales so that sphericity effects were of little significance. The sources of the phospholipid and additive compounds respectively were as follows: DOPC and PS, Lipid Products; benzo[a]pyrene (B-a-p), all-trans- and 13-cis-retinol, Sigma; 1,1bis(4-chlorobiphenyl)-2,2,2-trichlorethane (DDT), Aldrich, cholesterol, BDH (see Figure 1 for structures of these compounds). Solvents used were HPLC grade from Fisons. All experiments with the isomers of retinol were carried out under dim red light because of the light sensitivity of the compounds. Retinol working solutions were stored in the dark at -20 °C. The following monolayer systems were studied: DOPC; DOPC-0.4 PS; DOPC0.25 B-a-p; DOPC-0.3 all-trans- and 13-cis-retinol; DOPC-0.16 DDT; DOPC-0.26 cholesterol.
Results 1. ac Voltammograms. In Figure 2a, a differential capacity plot of the adsorbed DOPC layer is shown. The capacity of the potential region between -0.4 and -0.7 V (24) Nelson, A.; Benton, A. J. Electroanal. Chem. 1986, 202, 253270. (25) Oldham, K. B. J. Electroanal. Chem. 1983, 145, 9-20.
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Figure 2. Differential capacity (C) versus potential (-E) of the monolayer-coated mercury electrodes in electrolyte 0.1 mol dm-3 KCl with added 0.01 mol dm-3 phosphate buffer at pH 7.2. Differential capacity measured by out-of-phase (90°) voltammetry, scan rate 0.006 V s-1, frequency 75 Hz, and amplitude 0.0085 V. Arrows in diagram point to the differential capacity scales relevant to the individual plots. Monolayer: (a) DOPC, (b) DOPC-0.25 B-a-p (c) DOPC-0.3 13-cis-retinol. (a) (s) electrolyte only, (‚‚‚) electrolyte with added 4 × 10-4 mol dm-3 Tl(I) and (- - -) electrolyte with added 0.128 µmol dm-3 gramicidin and 4 × 10-4 mol dm-3 Tl(I). (b) and (c) (s) electrolyte with added 4 × 10-4 mol dm-3 Tl(I) and (- - -) electrolyte with added 0.128 µmol dm-3 gramicidin and 4 × 10-4 mol dm-3 Tl(I).
is 1.8 µF cm-2.24,26 At potentials more negative than -0.8 V reversible lipid reorientations are observed as two capacitance peaks. The presence of Tl+ in solution has only a small effect on the capacity-potential curve. On the other hand, when both Tl+ and gramicidin are added to the electrolyte, the capacity-potential plot is somewhat altered. There is an increase in capacitance at -0.4 V and the flat capacitance minimum is modified. The capacitance peaks are slightly depressed and broadened with Tl+ and gramicidin in the electrolyte. Modification of the adsorbed layer with bioactive compounds changes the form of the capacity-potential plot. As an example the capacity-potential plot of a DOPC-0.25 B-a-p monolayer coated electrode (Figure 2b) exhibits lower capacity minimum values, indicating a thicker monolayer and the capacity peaks are shifted to more negative potentials.23 The capacity-potential plots obtained at DOPC-0.3 13cis-retinol coated electrodes show a small lowering of the capacity minimum value and a modification of the capacity peaks (Figure 2c). The relative change of the low capacity-potential plots on addition of gramicidin and Tl+ to the solution is in the main similar to that of a pure DOPC layer except that the capacity minimum value is decreased. However the size of the capacitance increase at -0.4 V depends on the monolayer system and is highest in the example shown at the DOPC-0.3 13-cis-retinol coated electrode. 2. Cyclic Voltammograms and Potential Step Current-Time Plots. The voltammograms of Tl(I) in 0.1 mol dm-3 KCl at DOPC and DOPC-0.4PS coated electrodes are displayed in Figure 3. In the absence of gramicidin in the monolayer, Tl(I) is electroinactive. In the presence of gramicidin both reduction and oxidation peaks of Tl are observed. At the gramicidin-modified DOPC coated electrode (Figure 3A), there is a tendency for the reduction current peak to lose its peak-shaped characteristics with increase in scan rate but the reduction wave remains at a constant potential. The anodic peak shape is less affected by increase in scan rate and remains peak-shaped. At the gramicidin-modified DOPC-0.4 PS monolayer coated electrode (Figure 3B) voltammograms show increased current compared to the gramicidinmodified DOPC layer coated electrode.22 The potential (26) Moncelli, M. R.; Guidelli, R. J. Electroanal. Chem. 1992, 326, 331-338.
Nelson
Figure 3. Cyclic voltammograms (CVs) of 4 × 10-4 mol dm-3 Tl(I) in 0.1 mol dm-3 KCl with added 0.01 mol dm-3 phosphate buffer at pH 7.2. CVs shown in (b), (c), and (d) with 0.128 µmol dm-3 gramicidin added to the electrolyte. CVs carried out at (A) DOPC and (B) DOPC-0.4 PS coated electrodes. Scan rates used; (a and b) 0.2, (c) 2, and (d) 20 V s-1. Current scale refers to cyclic voltammograms depicted above it.
Figure 4. Cyclic voltammograms (CVs) of 4 × 10-4 mol dm-3 Tl(I) shown in (a), (c), (d), and (e), and of 4 × 10-4 mol dm-3 Cd(II) shown in (b), carried out in 0.1 mol dm-3 KCl with added 0.01 mol dm-3 phosphate buffer at pH 7.2 except where stated. CVs in (b), (c), (d), and (e) with added 0.128 µmol dm-3 gramicidin in electrolyte. CVs carried out at (A), DOPC-0.25 B-a-p and (B), DOPC-0.3 all-trans-retinol coated electrodes. Scan rates used: (a), (b), and (c) 0.2; (d) 2; (e) 20 V s-1. Dashed trace in (B),(e) depicts CV carried out in 0.05 mol dm-3 Mg(NO3)2 electrolyte with added 0.01 mol dm-3 acetate buffer at pH 5.8. Current scale refers to cyclic voltammograms depicted above it.
separation (∆Ep ) 0.57 V) between the cathodic and anodic peaks at scan rate 0.2 V s-1 is equivalent to that for a reversible reaction although increased peak potential separation is noted at higher scan rates. At all scan rates the voltammograms are peak-shaped. Sections A and B of Figure 4 show voltammograms of the reduction of Tl+ when B-a-p and all-trans-retinol,
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Langmuir, Vol. 12, No. 8, 1996 2061
Figure 5. Cyclic voltammograms (CVs) of 8 × 10-4 mol dm-3 Tl(I) at a DOPC-coated electrode with added 0.021 µmol dm-3 gramicidin in electrolyte shown in (a) and of 4 × 10-4 mol dm-3 Tl(I) at a DOPC-0.4 PS coated electrode with added 0.01 µmol dm-3 gramicidin in electrolyte shown in (b). Electrolyte, 0.1 mol dm-3 KCl with added 0.01 mol dm-3 phosphate buffer at pH 7.2. Scan rate, 20 V s-1.
respectively, are introduced into the DOPC monolayer, respectively. Tl+ is effectively electroinactive at electrodes coated with these monolayers. When these monolayers are modified with gramicidin, the voltammograms of Tl show increased current compared to the voltammograms obtained at a gramicidin-modified DOPC coated electrode. The reduction and oxidation peaks of Tl retain their peakshaped character, but their shape becomes rather altered at higher scan rates. In contrast to Tl+, Cd2+ is electroinactive at these gramicidin-modified coated electrodes similar to its behavior at the gramicidin-modified DOPC coated electrode.20 In the CVs of Tl carried out at a gramicidin-modified DOPC-0.25 B-a-P coated electrode (Figure 4A), the cathodic and anodic peaks become more separated at higher scan rates and the CVs appear rather drawn out. This is not evident in CVs carried out at a gramicidin-modified DOPC-0.3 all-trans-retinol coated electrode (Figure 4B) although at higher scan rates an increase in the current is noted at the beginning of the return sweep making the CV appear somewhat distorted. This distortion is removed when the cyclic voltammetry is carried out in Mg2+ electrolyte. CVs of Tl at gramicidinmodified DOPC-0.3 all-trans-retinol and DOPC-0.3 13cis-retinol coated electrodes in the same solution had precisely the same form and differed only slightly in their current values (not shown). In parts a and b of Figure 5 are displayed cyclic voltammograms of Tl at gramicidin-modified DOPC and DOPC-0.4 PS coated electrodes, respectively, with decreased concentrations of gramicidin in solution and in the monolayer with which it is in equilibrium. It is observed that with lower concentrations of gramicidin in the monolayer, the anodic peak shifts to more positive potentials leading to a wider potential separation between cathodic and anodic peaks (compare Figure 5a with Figure 3A,d and Figure 5b with Figure 3B,d, respectively). Apart from this effect there is no great change in the shape of the voltammogram. In Figure 6 are displayed plots of peak reduction current (ip) versus the square root of scan rate (v1/2) derived from the voltammograms of the various systems. These tend to a limiting value at high scan rate. It can easily be seen that the addition of compounds to the lipid monolayer influences the reduction current peak
Figure 6. Plots of reduction peak current (ip) versus square root of scan rate (v1/2) derived from cyclic voltammograms of 4 × 10-4 mol dm-3 Tl(I) in electrolyte with added 0.128 µmol dm-3 gramicidin at the following monolayer-coated electrodes: (a) (9) DOPC-0.16 DDT, (4) DOPC, (b) DOPC-0.25 B-a-p, (O) DOPC-0.3 13-cis-retinol, (2) DOPC-0.4 PS monolayer in 0.1 mol dm-3 KCl electrolyte with added 0.01 mol dm-3 phosphate buffer at pH 7.2; (b) (2) DOPC-0.25 B-a-p monolayer, (9) DOPC-0.3 13-cis-retinol monolayer in 1 mol dm-3 KNO3 electrolyte with added 0.01 mol dm-3 phosphate buffer at pH 7.2, (4) DOPC-0.25 B-a-p monolayer, (0) DOPC-0.3 13-cisretinol monolayer in 0.5 mol dm-3 Mg(NO3)2 electrolyte with added 0.01 mol dm-3 acetate buffer at pH 5.8.
height at the gramicidin-modified coated electrode throughout all the range of scan rates. In contrast to the effect of B-a-p and retinol additives in the gramicidin-modified monolayer, some chloroaromatic compounds, e.g., DDT, suppress the reduction current (see Figure 6a). Figure 6b shows quite clearly the influence of more concentrated K+ electrolyte compared to Mg2+ electrolyte in significantly decreasing the reduction currents at gramicidin-modified DOPC-0.25 B-a-P and DOPC-0.3 13-cis-retinol coated electrodes, respectively. Although experiments with Mg2+ electrolyte were carried out at a different pH to those in K+ electrolyte, there is no significant change in the reduction currents in the respective electrolytes within this pH range in the context of the results of this study.22 Some representative current-time plots from the potential step experiments are displayed in Figure 7. The currents recorded in these also depend on the lipid monolayer composition in the same way as the CV currents. At gramicidin-modified DOPC and DOPC-0.3 13-cis-retinol coated electrodes in 0.1 mol dm-3 KCl, the currents decreased significantly with increasing negative potential of the step. Discussion 1. Nature of the Adsorbed Layer. The structure of the phospholipid monolayer adsorbed on mercury has been described in past publications and consists of half a bilayer where the lipid tails sit next to the mercury surface and the lipid polar heads remain in solution.24 Tl electro-
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Figure 7. Current-time traces from potential step experiments. Potential steps from -0.2 V to (s) -0.6 V and (- - -) -0.7 V. 4 × 10-4 mol dm-3 Tl(I) in electrolyte with added 0.128 µmol dm-3 gramicidin at the following monolayer-coated electrodes and in the following electrolyte systems: (a) DOPC, (b) DOPC-0.4 PS monolayer; (c) DOPC-0.25 B-a-p; (d) DOPC0.3 13-cis-retinol in 0.1 mol dm-3 KCl electrolyte with added 0.01 mol dm-3 phosphate buffer at pH 7.2.
chemistry is observed between potentials -0.2 and -0.7 V where the lipid layer preserves this stable constant structure. At more negative potentials, the capacitance peaks correspond to lipid reorientations which are very sensitive to the interaction of the lipid with hydrophobic organic compounds.23 A change in shape and position of the capacitance peaks results from this which is specific to the interaction. When gramicidin is added to the solution, it partitions into the monolayer and forms helical water-filled channels. This modification of the capacitance-potential plot in the presence of Tl+ and gramicidin (Figure 2) corresponds to the transport of Tl+ through the gramicidin pore. The capacitance increase at -0.4 V represents part of the Tl+/Tl(Hg) redox pseudocapacitance peak and is highest in the case where the reduction currents are largest at the gramicidin-modified DOPC0.3 13-cis-retinol coated electrode. All work reported in this study refers to a fully covered electrode in which the gramicidin channel maintains a position normal to the lipid layer surface. Any tilting of the channel from the layer normal by expanding the mercury drop leads to a decrease in the reduction current at the coated electrode. Accordingly, full coverage of the electrode was always checked before each CV experiment by capacitance measurements of the adsorbed layer. 2. Electrochemistry of Tl at the GramicidinPhospholipid Coated Mercury Electrode. 2.1. Model. Figure 8a displays a model of the phospholipidgramicidin coated mercury electrode. As an initial first assumption, the fraction of electrode exposed to channeloccluded water due to the presence of gramicidin, (1 - θ), is taken to be greater than 0.01. The average distance between the pores, d, is calculated from the relation given by Amatore et al.27 for a blocked electrode covered in diskshaped electroactive sites
d ) 2r(1 - θ)-1/2
(1)
which in this case is >40 Å, where r is the radius of the gramicidin pore which is 2 Å.8 The model in Figure 8a for (27) Amatore, C.; Saveant, J. M.; Tessier, D. J. Electroanal. Chem. 1983, 147, 39-51.
Figure 8. (a) Simplified model of the distribution of the gramicidin monomolecular channels in a section of phospholipid monolayer (B) sandwiched between the two conducting phases electrolyte (A) and mercury electrode (C) respectively. d denotes the average distance between the pore centers, 2r is the pore diameter and M is the pore length. (b) Schematic diagram of the potential energy profile of a cation passing through the monomolecular gramicidin channel (in the phospholipid monolayer (B)) from the electrolyte (A) to the mercury (C) phase. WTotal is the sum of the repulsive dielectric work, WE, and mostly attractive energy of interaction, WChem, between the cation and the polar groups of the polar wall. M is the pore length. The nature of the lipid environment influences both these energy terms.
the gramicidin distribution is supported by previous work. This has shown that strong peptide-lipid interactions are favored between the four tryptophan residues of the gramicidin channel mouth and the lipid polar groups through hydrogen bonding.28 As a consequence a dispersion of conducting monomer gramicidin channels is favored in the lipid phase when the gramicidin partitions from the very low concentrations of gramicidin in the aqueous phase.12 In addition the known lateral mobility of the gramicidin8 within lipid layers will lead to the random distribution of the monomer channels. In this study the maximum scan rate used in cyclic voltammetry is 20 V s-1 (time scale ∼1.28 ms), which gives rise to a diffusion layer thickness of ∼(DRT/Fv)1/2 ) 16000 Å, where D is the diffusion coefficient of the Tl+ ion which is 10-4.7 cm2 s-1.29 It is noted that the diffusion layer thickness is 102.6 times in excess of the average distance between the pores. As a result, in the shortest time scale of the experiment, at this assumed minimum value of (1 - θ), the major fraction of the diffusion of the ion within the diffusion layer to the channel openings can be considered as linear. The model of Amatore et al.27 can be further applied to the system in Figure 8a. These authors have predicted the shapes of cyclic voltammograms obtained from electrochemical studies at such electrode systems in the form (28) Hu, W.; Lee, K.-C.; Cross, T. A. Biochemistry 1993, 32, 70357047. (29) Kolthoff, M.; Lingane, J. J. Polarography; Interscience Publishers: New York, 1952; Vol. 1, p 95.
Ion Channel Transport
of zone diagrams as a function of r, v, (1 - θ), and the heterogeneous rate constant of the electroactive ion’s redox couple at the uncoated electrode, ks,oapp ()1.8 cm s-1 for Tl+/Tl(Hg30). According to this model, in the phospholipid-gramicidin system on the mercury electrode under the conditions specified above, at the lower scan rate of 0.2 V s-1 a reversible cyclic voltammogram should be observed as shown in the top right-hand corner of this zone diagram (Figure 3 of ref 27) with no suppression of peak heights. Increase of scan rate or a decrease in (1 θ) will bring about an increase in the potential separation of the peaks and tendency to quasi-reversibility. As a result, the effect of discrete active sites or pores with the dimensions and distribution given above on a blocked electrode does not significantly influence the voltammetry which should behave almost exactly like that on an uncoated electrode. The approach described above does not take into account the passage of the ion through the channel which if it involves some energy barrier will become rate limiting since the rate constant for the Tl+/Tl(Hg) process is fast. Within the bimolecular gramicidin channel there are generally considered to be three energy barriers to the passage of ions.15-19,31 There are energy barriers at either end of the channel corresponding to the desolvation and solvation of the ion consequent with its entry into and exit from the channel respectively (e.g., ref 31). There is also an electrostatic energy barrier within the channel corresponding to the image forces due to the presence of a low dielectric medium surrounding the channel. Between the two energy barriers in each half of the bimolecular gramicidin channel there exists a binding site for the translocating ion. In the unimolecular gramicidin channel in the system in this study, the energy barriers are asymmetrically placed. The central energy barrier of the bimolecular channel is split in two, and half the barrier is directly apposed to the mercury surface. Figure 8b depicts the energy barrier structure to ion translocation of the gramicidin channel in the monolayer on the mercury surface. The energy well at the channel base has not been drawn but is represented by a gap. At this point the ion exits the channel and is subsequently reduced on the mercury surface. The height and exact position of the energy barrier within the channel are influenced by the membrane potential,17 the dimensions of the channel mouth,15 and the lipid layer composition, factors which affect the image forces (WE) acting on the ion within the pore. Interactions between the polarizable peptide groups of gramicidin and the translocating ion influence the chemical energy (WChem) of the ion. Both terms affect the heights of the energy barriers to ion translocation. For channel systems in low dielectric lipid media, an applied potential field would extend over the whole channel protruding into the solution.15 As a result, the translocation of the ion over the energy barrier apposed to the mercury surface will be influenced by only a small fraction of the total applied field. In the system under consideration in this study the rate limiting step from the binding site within the channel over the energy barrier to the mercury surface can be compared to a chemical process prior to the reversible reduction of Tl+ (CE process). In addition to the effects due to image forces and chemical interactions within the pore, the transport of ions within the pore will be affected by events occurring at the mouth of the pore. In this way the passage of ions within the pore will be influenced by the electrolyte composition. (30) Galus, Z. Fundamentals of Electrochemical Analysis; Ellis Horwood: Chichester, 1976; pp 37, 67, 259, 292. (31) Hille, B. Ionic Channels of Excitable Membranes; Sinauer Associates: Sunderland, MA, 1984; pp 291-314, 289.
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2.2. Cyclic Voltammetry. In the CE process in cyclic voltammetry, the height of the reduction wave tends to a limiting current, ik, as the time scale of the experiment is decreased given by30,32-35
ik ) FAC*k1µ ) FAC*K(k1 + k2)1/2D1/2
(2)
where A is the electrode area, C* is the concentration of electroinactive substance in solution prior to the chemical step, k1 is the first-order forward rate constant governing the chemical step, K ) k1/k2 is the equilibrium constant of the chemical step, and µ is the reaction layer thickness. This equation can be applied to the ion transport across a channel-modified monolayer. In this case, the “pure kinetic current” represents the rate limiting flux of Tl+ ions across the monolayer of thickness, M, under steady state conditions. Indeed in this case
ik ) FACmk1M
(3)
where the thickness of the hydrocarbon region of the monolayer, M, is assumed to be the same as the length of the channel given as 10-6.9 cm8 and Cm is the concentration of the ion at the mouth of the pore assumed to be equivalent to the bulk concentration value. Conceptually ik can be viewed as a steady state current attained at sufficiently short experimental time scales where the rate of translocation of the ion across the channel-modified monolayer is exactly compensated by the diffusion of the ion toward the monolayer surface. k1 is the first-order permeability rate constant associated with the transport of the ion across the channel-modified monolayer expressed in flux of Tl+ per unit volume of monolayer per unit concentration of Tl+ in solution. It is to be noted that k1M ) kp where kp is the heterogeneous permeability coefficient or permeability associated with the transport of Tl+ across the monolayer expressed as flux of Tl+ per unit area of monolayer per unit concentration of Tl+ in solution.20,22 As a result if the rate limiting process involves some energy barrier in the passage of the ion through the channel, k1 corresponds to the product of the first-order rate constant, kt, governing the translocation rate of the ion within the pore and the fraction (1 - θ) of electrode exposed to the channel openings due to the presence of gramicidin; i.e.
k1 ) (1 - θ)kt
(4)
The first-order rate constant describing gramicidin channel translocation of univalent ions is variously reported but the figure generally quoted is that for Na+ crossing the central energy barrier of the bimolecular gramicidin channel an average value of which is 1.5 × 107 s-1.36 If a value of (1 - θ) of 0.01 is assumed, values of k1 as measured in the system under consideration in this study should be ∼1.5 × 105 s-1. Experimental evidence from the cyclic voltammogram data tends to conform to the models proposed in Figure 8 by virtue of the following points. The dispersion of monomer gramicidin channels within the lipid monolayer as displayed in the model in Figure 8a is commensurate with the regular increase of the permeability properties to Tl+ of the gramicidin-modified monolayer in a curvi(32) Nicholson, R. S.; Shain, I. Anal. Chem. 1964, 36, 707-723. (33) Saveant, J. M.; Vianello, E. Electrochim. Acta 1963, 8, 905-923. (34) Bard, A. J.; Faulkner, L. R. Electrochemical Methods: Fundamentals and Applications; John Wiley and Sons: New York, 1980; p 445. (35) Bottempelli, G.; Magno, F.; Mazzocchin, G. A.; Seeber, R. Ann. Chim. 1989, 79, 103-216. (36) Cooper, K. E.; Gates, P. Y.; Eisenberg, R. E. J. Membr. Biol. 1988, 106, 95-105.
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linear manner to a limit with increase in gramicidin in the electrolyte solution (see Figure 5 of ref 20). At the same time, there is no abrupt change in the shape of the voltammograms with increase in gramicidin in solution (compare Figure 5 with Figure 3). The presence of clusters or islands of gramicidin molecules in the lipid monolayer could manifest itself as discontinuities in the permeability properties of the gramicidin-modified monolayer and thus the Tl voltammetry as the gramicidin concentration in the solution was increased. Examination of the cyclic voltammograms in Figures 3 and 4 shows that those of the system Tl(I)/Tl(Hg) at the gramicidin-modified DOPC-0.4 PS and the DOPC-0.3 retinol coated electrodes most nearly resemble those predicted from the model described in Figure 8a since at lower scan rates they are almost completely reversible. The shape of the voltammogram at all scan rates conforms to that observed in the top right-hand corner of Figure 3 of ref 27 where there is an overlapping of the diffusion spheres to each channel opening indicating that nonlinear diffusion is not significant in the time scale of the experiment.37 At higher scan rates there is some suppression of the reduction peak current and separation of the peak potentials. In addition, with the value of ks,oapp used in the above model, the earlier assumptions of gramicidin coverage are within a reasonable range, since if surface concentrations of gramicidin are lower, the peak potentials separate as shown in Figure 5b. In any case a minimum gramicidin coverage, (1 - θ), of 10-3.26 has been calculated from the following relationship27
(1 - θ)-1 E (DRT/Fv)1/2/4.44r
(5)
This is commensurate with the observed CVs of welldefined cathodic and anodic peaks at the maximum scan rate, v, of 20 V s-1. The voltammograms obtained at other monolayer-coated electrodes show a greater suppression of the peaks which is accentuated at higher scan rates in which the reduction peak current tends to a limiting value. In all CVs there is an absence of a sigmoidal shape of the voltammograms and the presence of a well-developed anodic peak which shifts to more positive potentials with a lower concentration of gramicidin in the monolayer (see Figure 5). This suggests that radial diffusion is not important in contributing to the electrochemical behavior.37 Additional evidence that the CV s do not fit the model for a nonlinear diffusion mechanism is shown by the absence of negative shifts of the half-peak potential, E1/2, of the reduction peak from the formal electrode potential, E° ()-0.433 V for Tl(I)/Tl(Hg)), in the CVs where the peak current approaches a limiting value (see Figure 3A). Amatore et al.27 have derived the following equation from their model in the situation of nonlinear diffusion of electroactive ions to an array of individual active sites widely separated from one another
ks,oapp ) (D/0.6r) exp[(RF/RT)(E1/2 - E°)]
(6)
If R is approximated as 0.5, a potential shift, ∆E ()E1/2 - E°), of -0.356 V is predicted. In the case of the value of ks,oapp being increased 40-fold from 1.8 to 72 cm s-1 as has been observed in other channel systems on electrodes,38 a ∆E value of -0.163 V is still expected. The cyclic voltammograms of the systems in this study where the reduction current is suppressed show greater resem(37) Cheng, I. F.; Whiteley, L. D.; Martin, C. R. Anal. Chem. 1989, 61, 762-766. (38) Bilewicz, R.; Majda, M. J. Am. Chem. Soc. 1991, 113, 54645466.
blance to those of a CE process in which the cathodic peak loses its peak-shaped character at higher scan rates and the anodic peak is always well defined.30,32-35 The results of this study are analyzed using the following Nicholson and Shain equation which has been applied successfully to derive rate constants from electrochemical reactions involving a CE mechanism32
ip/id ) 1/[1.02 + 0.471(a1/2/K(k1 + k2)1/2]
(7)
where id is the reduction current observed at an uncoated electrode in the absence of any complicating reactions and a ) Fv/RT. Equation 7 can be rewritten and applied to Tl(I) reduction at the gramicidin-modified monolayer coated electrode
ip/id ) 1/[1.02 + 0.471(a1/2D1/2/kp)]
(8)
where kp/D1/2 ) K(k1 + k2)1/2.30 Rearrangement of eq 8 shows that by plotting a1/2ip-1 versus a1/2 for the various systems in Figure 9, a straight line is obtained, the intercept of which should be 1.02/(ida-1/2).35 The theoretical value of this intercept can be calculated. For the reversible Tl(I)/Tl(Hg) process, ida-1/2 ) 0.4463FACD1/2, which is a constant value and with the experimental conditions as reported in this study, ida-1/2 ) 0.6673 × 10-6 A s1/2 or the theoretical value of the intercept in Figure 9 is 1.506 × 106 A-1 s-1/2. When the value of ida-1/2 is calculated from the intercept and substituted into the slope of the graph, ∆(a1/2ik-1)/∆a1/2 ()0.471/kpD-1/2ida-1/2), kpD-1/2 can be estimated. Observation of Figure 9 shows that generally the voltammetric results from the systems studied in this work show a good linear fit in the plots displayed. With some exceptions, the intercept is within (15% of the theoretical value. Higher values of the intercept can be observed in the plots derived from the voltammograms obtained with the systems of gramicidin-modified DOPC0.25 B-a-p coated electrodes in 0.1 mol dm-3 KCl and of gramicidin-modified DOPC coated electrodes in 0.5 mol dm-3 Mg(NO3)2, respectively. In the former system the points do not fit the linear plot so well but exhibit a curvilinear plot. Values of kp and k1 calculated from several systems investigated are displayed in Table 1. The results in Table 1 show the effects of PS, retinol, and cholesterol in increasing and DDT in decreasing the apparent permeability of the gramicidin-modified monolayer. They also show the influence of high K(I) electrolyte ion concentration but not Mg(II) in decreasing the gramicidin-modified monolayer permeability regardless of the monolayer system. The values of k1 fall on either side of the earlier predicted value of k1 for this system of 1.5 × 105 s-1. 2.3. Chronoamperometry. The chronoamperometry of redox couples at blocked electrodes with active sites39,40 and at ensembles of microelectrodes41 has been previously modeled39-41 and studied experimentally39,40 and in some cases the treatment of the problem is similar to that used for a CE mechanism.39 However using the treatment in ref 41 together with the minimum value of (1 - θ) of 10-3.26 estimated earlier, the time when the diffusion spheres begin to overlap is 2 × 10-5 s and is not significant within the time scale of the potential step experiment. In this study the results of the potential step experiments were used firstly to show the potential dependence of the current and secondly to substantiate the values of (39) Gueshi, T.; Tokuda, K.; Matsuda, H. J. Electroanal. Chem. 1978, 89, 247-260. (40) Bizzotto, D.; McAlees, A.; Lipkowski, J.; McCrindle, R. Langmuir 1995, 11, 3243-3250. (41) Scharifker, B. J. J. Electroanal. Chem. 1988, 240, 61-76.
Ion Channel Transport
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Figure 9. Plots of (Fv/RT)1/2 × reciprocal peak current (a1/2ip-1) versus a1/2 derived from cyclic voltammograms of 4 × 10-4 mol dm-3 Tl(I) in electrolyte with added 0.128 µmol dm-3 gramicidin at the following monolayer-coated electrodes and in the following electrolyte systems: (a) (9) DOPC-0.16 DDT, (4) DOPC, (b) DOPC-0.25 B-a-p, (O) DOPC-0.3 13-cis-retinol, and (2) DOPC-0.4 PS monolayer in 0.1 mol dm-3 KCl electrolyte with added 0.01 mol dm-3 phosphate buffer at pH 7.2; (b) (b) DOPC in 0.1 mol dm-3 KCl electrolyte, (9) DOPC and (4) DOPC-0.3 13-cis-retinol monolayer in 1 mol dm-3 KNO3 electrolyte with added 0.01 mol dm-3 phosphate buffer at pH 7.2, and (0) DOPC and (2) DOPC-0.3 13-cis-retinol monolayer in 0.5 mol dm-3 Mg(NO3)2 electrolyte with added 0.01 mol dm-3 acetate buffer at pH 5.8.
the rate constant determined above by the analysis of the CV results. The plots of the current (i) versus the semiintegral of the current (m) derived from the current-time traces are displayed in Figures 10 and 11. As given by Oldham25 these plots should be linear for a CE electrode process obeying the equation
i ) i0 - zm
(9)
where i0 is the current at t ) 0 and is equivalent to the previously defined ik in CV. Figures 10 and 11 show that in almost all cases within the time scale of the experiment the plots can be extrapolated to the i axis to give the value of i0. Theoretically under the experimental conditions used in this study, i0z-1, which is the intercept on the m axis, should be equal to FCmAD1/2 25 or 1.52 µA s1/2 assuming that Cm is approximately equal to the bulk Tl(I) concentration value. In fact the intercept always falls short of this value. The reasons for this are associated with the behavior of the Tl+ ion within short time scales and are currently under investigation. The results in Figures 10 and 11 show the effect of K+ in the electrolyte on the transport of Tl+ within the pore whereby the value of the current decreases when the potential step is more negative. This is most evident in the results at the gramicidin-modified DOPC-0.3 13-cisretinol coated electrode in KCl electrolyte (Figure 10b and
Figure 11d) and accounts for the distorted nature of the CVs at this coated electrode system at high scan rates (see Figure 4B,e). An explanation for this result is that the increased potential field attracts the K+ ion into the mouth of the pore and this effect interferes with the translocation of Tl+ through the channel. Supporting evidence for this interpretation is that the current value is even further decreased in more concentrated K+ electrolyte and this effect is absent when the experiments are carried out in Mg2+ electrolyte (see Figure 11). Values of rate constants calculated from i0 ()FACmkp(i0)) on the basis that Cm is equivalent to the bulk concentration of Tl+ are displayed in Table 1. In most cases, rate constants were estimated from the -0.2 to -0.6 V potential step current-time traces. Generally the value of kp(i0) agrees with that of kp(CV) to within about 25%. However in the presence of Mg2+ electrolyte at the DOPC-coated electrode a lower calculated value of kp(i0) than kp(CV) by a factor of 2 is obtained presumably due to a lower value of Cm than in the bulk solution. A decreased value of Cm arises from a lowered Tl(I) concentration on the surface of the monolayer which results from the fixed charge effects due to adsorption of Mg2+ on the lipid surface as discussed in earlier papers.22,42 Similar to the voltammetric results, the system of reduction of Tl(I) at gramicidin-modified DOPC-0.25 B-a-p coated electrodes is more complicated than the other systems investigated since the i versus m plots derived from the current-time traces obtained for the reduction of Tl(I) in dilute electrolyte (see Figure 10d) show definite curvature. 3. Gramicidin-Mediated Permeability of the Monolayer Systems. The variation of the gramicidinmediated permeability of the monolayer to Tl+ as a function of the lipid and solution environment could be due to alterations in a number of variables as follows: (a) the gramicidin concentration in the monolayer; (b) the distribution and structure of gramicidin in the monolayer; (c) the height and significance of the energy barriers within the channel to ion transport according to the model in Figure 8b. Changes in the gramicidin coverage in the layer may be a factor in the effect of additive compounds within the layer and in the influence of electrolyte composition and concentration on the gramicidin-mediated permeability but it appears not to be of primary significance in this study. One reason for this is that the incorporation of additive compounds into the layer would be expected to lower the gramicidin coverage when the layer becomes more compact but in most cases the current increases. In the case of the effect of B-a-p and retinol on Tl electrochemistry at gramicidin-modified phospholipid-coated electrodes, these compounds do not increase the gramicidin-mediated permeability of the monolayers by widening the radius of the channel since the channel remains impermeable to Cd2+ ions (see Figure 4) and is not blocked by Mg2+ electrolyte ions. Gross structural effects on the gramicidin channel in the presence of these compounds within the monolayer are also unlikely since the conversion between the conducting and nonconducting forms of gramicidin at room temperature is slow10,11 and is dependent on the solvent history of the gramicidin. It is therefore proposed that the variation of the gramicidinmediated permeability of the monolayer due to different monolayer composition is predominantly caused by a change in the height and significance of the energy barriers within the channel to ion transport. It is initially assumed that the largest energy barrier to ion translocation is (42) Meijer, L. A.; Leermakers, F. A. M.; Nelson, A. Langmuir 1994, 10, 1199-1206.
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Table 1. Values of First-Order Rate Constant, k1, and Heterogeneous Permeability Coefficient, kp Deriveda from the Reduction of 4 × 10-4 mol dm-3 Tl(I) in Electrolyteb with Added 0.128 µmol dm-3 Gramicidin at the Phospholipid Monolayer-Additive Coated Electrode monolayer/DOPCmole fraction additive
electrolyte, concn mol dm-3
k1(CV)/105 s-1
kp(CV)
kp/10-2 cm s-1 -E/V
kp(i(0))
DOPC DOPC DOPC-0.4 PS DOPC DOPC-0.3 13-cis-retinol DOPC-0.3 13-cis-retinol DOPC-0.3 13-cis-retinol DOPC-0.16 DDT DOPC-0.26 cholesterol
KCl, 0.1 KNO3, 1.0 KCl, 0.1 Mg(NO3)2, 0.5 KCl, 0.1 KNO3, 1.0 Mg(NO3)2, 0.5 KCl, 0.1 KCl, 0.1
0.92 0.72 4.64 1.61 3.85 0.64 3.8 0.50 1.60
1.15 0.91 5.84 2.03 4.84 0.81 4.78 0.64 2.02
0.6 0.7 0.6 0.6 0.6 0.6 0.6 0.6 0.6
1.02 0.69 6.76 1.04 5.84 0.57 4.50 0.62 2.11
a k (CV) and k (CV) determined from CV experiments and k (i ) determined from potential (-E/V indicated) step experiments as 1 p p (0) described in the text. b K+ electrolyte buffered at pH 7.2 with 0.01 mol dm-3 phosphate and Mg2+ electrolyte buffered at pH 5.8 with 0.01 mol dm-3 acetate.
Figure 10. Plots of current (i) versus the semi-integrated current derived from current-time traces of potential step experiments. Potential steps from -0.2V to (4) -0.6 V and (b) -0.7 V. 4 × 10-4 mol dm-3 Tl(I) in 0.1 mol dm-3 KCl electrolyte with added 0.01 mol dm-3 phosphate buffer at pH 7.2 and 0.128 µmol dm-3 gramicidin at the following monolayer-coated electrodes: (a) DOPC-0.4 PS monolayer; (b) DOPC-0.3 13cis-retinol; (c) DOPC-0.26 cholesterol; (d) DOPC-0.25 B-a-p; (e) DOPC; (f) DOPC-0.16 DDT. Best fit lines (dashed trace) are drawn through the plots derived from potential step experiments to -0.6 V.
within the channel and is due to dielectric or image forces from the surrounding low dielectric hydrocarbon chains of the monolayer. Accordingly the presence of negatively charged lipids (PS) in the monolayer increases the rate of ion transport since a dipolar monolayer potential is predicted to lower this energy barrier significantly.17 Presumably the π-electron-rich retinol and B-a-p have individually strong effects due to their greater polarizability compared to the hydrocarbon chains of the lipid. These compounds may also directly interact with the ion by long range associations between the π-electrons of these molecules and the ions within the pore.43 At the same time aromatic and polyconjugated additive compounds could associate with the tryptophan residues of the β-helix of gramicidin situated at the mouth of the channel thus (43) Kumpf, R. A.; Dougherty, D. A. Science 1993, 261, 1708-1710.
Figure 11. Plots of current (i) versus the semi-integrated current derived from current-time traces of potential step experiments. Potential steps from -0.2 V to (4) -0.6 V and (b) -0.7 V. 4 × 10-4 mol dm-3 Tl(I) in electrolyte with added 0.128 µmol dm-3 gramicidin at the following monolayer-coated electrodes and in the following electrolytes respectively: (a) DOPC, (c) DOPC-0.3 13-cis-retinol, (e) DOPC-0.25 B-a-p monolayer in 0.5 mol dm-3 Mg(NO3)2 with added 0.01 mol dm-3 acetate buffer at pH 5.8 and (b) DOPC, (d) DOPC-0.3 13-cisretinol, and (f) DOPC-0.25 B-a-p monolayer in 1 mol dm-3 KNO3 electrolyte with added 0.01 mol dm-3 phosphate buffer at pH 7.2. Best fit lines (dashed trace) are drawn through the plots derived from potential step experiments to -0.6 V except in (b) where the potential step is to -0.7 V.
influencing the dipole moment of these residues. The dipole moment of the tryptophan residues has been implicated as important in controlling the conductance of ions in the channel.28,44 Accordingly any change in this dipole moment from interaction with additive compounds would affect ion transport through the channel. Thickening of the monolayer also has an effect of increasing the rate constant of translocation as is illustrated by the effect of cholesterol which is known to thicken phospholipid monolayers.45 The compounds which thicken the layer (44) Becker, M. D.; Greathouse, D. G.; Koeppe, R. G.; Anderson, O. S. Biochemistry 1991, 30, 8830-8839. (45) Nelson, A.; Auffret, N. J. Electroanal. Chem. 1988, 244, 99113.
Ion Channel Transport
will stress the monolayer-gramicidin junction and thus alter the channel mouth structure. An increase in the channel mouth size has been predicted as decreasing the energy barrier between the channel constriction and the bimolecular channel center or unimolecular channel base in this instance. This increases the channel translocation rate after channel entry. A similar effect could operate for B-a-p, which also thickens the monolayer,23 on the gramicidin channel transport. Conclusions 1. In the system of ion channels within a phospholipid layer investigated in this study, experimental evidence tends to support the proposed model that nonlinear diffusion effects are not significant in the electrochemistry of the probe ion, Tl+, within the time scale of the experiment. The electrode process has been modeled as a CE mechanism where the rate limiting step is the translocation of the ion within the channel. K+ electrolyte ion interferes with the Tl+ translocation process in being drawn into the pore by the applied field and at high K+ electrolyte ion concentration, the permeability of all gramicidin-modified monolayer-coated electrodes to Tl(I) is suppressed.
Langmuir, Vol. 12, No. 8, 1996 2067
2. Many hydrophobic additives in the lipid layer, in particular some polyaromatic and polyconjugated compounds, selectively alter the permeability of gramicidinmodified DOPC monolayers to Tl+. Up to a 5-fold increase in gramicidin-mediated permeability is noted in monolayers with added retinol. This increase is of the same order as that seen in the presence of a similar concentration of negatively charged lipids (PS) in the layer. The chlorinesubstituted aromatic pesticide DDT within the gramicidinmodified monolayer causes a significant depression in the apparent permeability. 3. It is proposed that the dependence of permeability to Tl+ on additives in the layer is predominantly caused by the effect of the compounds within the monolayer on the energy barrier to the translocation of the ion within the channel. These compounds can alter the image forces acting on the ion passing through the channel as well as directly interacting with the ion itself. The results of the study show that bioactive compounds can selectively influence the gramicidin channel function through interacting with a phospholipid-gramicidin system. LA9507850
