Influence of Fixed Charge and Polyunsaturated Compounds on the

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Langmuir 1997, 13, 5644-5651

Influence of Fixed Charge and Polyunsaturated Compounds on the Monomolecular Gramicidin Channel Function in Phospholipid Monolayers: Further Studies Andrew Nelson Plymouth Marine Laboratory, Citadel Hill, Plymouth PL1 2PB, U.K. Received January 9, 1997. In Final Form: July 14, 1997X An investigation into the effect of adsorbed and incorporated fixed charge and incorporated polyunsaturated compounds in dioleoylphosphatidylcholine (DOPC) monolayers on the translocation of Tl+ through the monomolecular gramicidin channel is reported in this paper. The work also includes a study of the interaction of the tricyclic neuroleptic drug chlorpromazine with DOPC monolayers and the influence of chlorpromazine on the transport of Tl+ through the gramicidin channel. Techniques used were out-of-phase AC voltammetry and cyclic voltammetry and chronoamperometry of the Tl(I)/Tl(Hg) couple. It was shown that negatively charged phosphatidylserine (PS) when incorporated into DOPC layers increases the gramicidin-mediated permeability of the layers by increasing the concentration of Tl+ on the monolayer surface and increasing the rate of translocation of the ion in the channel. Positively charged ions which adsorb onto lipid layers have the opposite effect. Polyunsaturated compounds when incorporated into DOPC monolayers have influences on gramicidin-mediated Tl+ translocation similar to those of negative fixed charge. The chlorpromazine interaction with DOPC layers and its influence on the transport of Tl+ in the pore depends on its charge. At high solution pH above its pKa of 9.3 its effect on the gramicidin-mediated permeability is similar to that of a polyunsaturated compound. At low solution pH its positive charge dominates its influence on the transport of Tl+ in the channel, decreasing the gramicidin-mediated permeability.

Introduction The interaction of biologically active compounds with cell membranes is always of importance (e.g. refs 1-5). The reason for this is that these compounds can selectively modify or disrupt membrane structure and alter the functional properties of enzymes and ion channels located within the membrane. These effects can be studied with model systems which provide more information on the mechanisms involved under controlled conditions. It has been shown for example by using the mercury-adsorbed phospholipid monolayer membrane model6,7 that bioactive compounds not only selectively interact with the lipids but also influence the functioning of model ion channels situated within the monolayer.8 In a previous study,8 both cyclic voltammetry and chronoamperometry were used as experimental techniques to probe the transport of Tl+ through the monomolecular gramicidin channel in a phospholipid monolayer in the presence of a number of bioactive compounds. The subsequent analysis was based on the justified model that radial diffusion effects9-12 were not significant within the time scale of the experiment. As a result, the process was modeled as a CE mechanism (chemical step preceding electron transfer), where the ratecontrolling process was the passage of the ion through the X Abstract published in Advance ACS Abstracts, September 1, 1997.

(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) Miller, I. R. In Topics in Bioelectrochemistry and Bioenergetics; Milazzo, G., Ed.; John Wiley and Sons: Chichester, 1981; Vol. 4, pp 161-224. (7) Nelson, A.; Benton, A. J. Electroanal. Chem. 1986, 202, 253-270. (8) Nelson, A. Langmuir 1996, 12, 2058-2067. (9) Amatore, C.; Saveant, J. M.; Tessier, D. J. Electroanal. Chem. 1983, 147, 39-51. (10) Cheng, I. F.; Whitely, L. D.; Martin, C. R. Anal. Chem. 1989, 61, 762-766. (11) Bilewicz, R.; Majda, M. J. Am. Chem. Soc. 1991, 113, 54645466. (12) Scharifker, B. J. J. Electroanal. Chem. 1988, 240, 61-76.

S0743-7463(97)00030-9 CCC: $14.00

Figure 1. Structures of (A) benzo[a]pyrene, (B) all-transretinol, and (C) chlorpromazine.

channel. It was shown that monolayer surface charge and monolayer and electrolyte composition strongly influenced ion transport through the channel.8 This present investigation is focused on identifying more closely the mechanisms involved which give rise to these observed effects. In the first part of this work, various well characterized model systems of dioleoylphosphatidylcholine (DOPC) monolayers with adsorbed fixed charge and incorporated additive compounds have been examined. The aim was to elucidate the means by which the charge structure and the lipid environment of phospholipid monolayers influence the translocation of ions in the incorporated gramicidin channels. The translocation of Tl+ across gramicidin-modified monolayers was investigated by studying Tl(I) reduction current transients in response to potential steps. The lipid environment was altered by adding the polyaromatic benzo[a]pyrene and the polyconjugated all-trans-retinol (see Figure 1) to the monolayer. The fixed positive charge on the monolayer surface was varied by carrying out experiments in Mg2+ © 1997 American Chemical Society

Monomolecular Gramicidin Channel Function

electrolyte, which adsorbs on DOPC layers,13,14 and in K+ electrolyte with added Dy3+, which also adsorbs on DOPC layers.13,14 To incorporate negative fixed charge in the monolayer, the phospholipid phosphatidylserine (PS), which is negatively charged at pH 7,15 was added to the DOPC monolayer. In the second part of the study a specific model system consisting of a DOPC monolayer in contact with the drug chlorpromazine (see Figure 1) was examined. The objective was to understand the interaction between chlorpromazine and DOPC and the manner in which the chlorpromazine in the DOPC layer affects the gramicidin channel transport of ions. Measurements of capacitance were done to qualitatively assess the interaction of chlorpromazine with the DOPC layer. This is possible, since an electrode which is fully covered with a pure DOPC monolayer possesses a characteristic capacity minimum value of > k1 and kp has precisely the same meaning as in the analysis of the potential step current transients. Rearrangement of eq 78,32 shows that, by plotting a1/2ip-1 versus a1/2 for the various systems, a straight line is obtained, the intercept of which should be 1.02/(ida-1/2). The theoretical value of this intercept can be calculated with the experimental conditions reported

(27) Galus, Z. Fundamentals of Electrochemical Analysis; Ellis Horwood: Chichester, 1993; pp 236 341, and 368. (28) Bard, A. J.; Faulkner, L. R. Electrochemical Methods: Fundamentals and Applications; John Wiley and Sons: New York, 1980; pp 507, 513, and 166. (29) Oldham, K. B. Anal. Chem. 1986, 58, 2296-2300.

(30) Kolthoff, M.; Lingane, J. J. Polarography; Interscience Publishers: New York, 1952; Vol. I, p 95. (31) Nicholson, R. S.; Shain, I. Anal. Chem. 1964, 36, 707-723. (32) Bottempelli, G.; Magno, F.; Mazzochin, G. A.; Seeber, R. Ann. Chim. 1989, 79, 103-216.

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Figure 3. Plots of current (i) versus the semi-integrated current (m) derived from current-time transients from potential step experiments of time period to 0.025 s and 0.3 s, respectively. A best fit line is drawn through the longer time scale plots. In part A the intercepts of this line on the i axis (i0) and the m axis (i0β-1) and its slope (-β) are labeled. Potential steps are from -0.2 V to (O) -0.6 V and to (f) -0.7 V. Solutions are 4 × 10-4 mol dm-3 Tl(I) in electrolyte with 0.01 mol dm-3 buffer and 0.128 µmol dm-3 gramicidin with the following monolayer-coated electrode and electrolyte systems: (A) DOPC in 0.1 mol dm-3 KCl electrolyte with phosphate buffer at pH 7.2; (B) DOPC-0.4 PS in 0.1 mol dm-3 KCl electrolyte with phosphate buffer at pH 7.2; (C) DOPC in 0.1 mol dm-3 KCl and 10-3 mol dm-3 Dy3+ with acetate buffer at pH 5.3; (D) DOPC-0.25 B[a]p in 0.1 mol dm-3 KCl electrolyte with phosphate buffer at pH 7.2.

in this study as 1.51 × 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, ∆(a1/2ip-1)/∆a1/2 ()0.471/ kpD-1/2ida-1/2), kp/D1/2 can be estimated. Results Figure 2 shows examples of the current versus time plots for four of the systems studied where the potential was stepped to -0.6 V and both the short and long time scale data are combined. As seen previously, the additives B[a]p and retinol considerably increased the currents across the gramicidin-modified layer8 whereas positive fixed charge as Dy3+ ions adsorbed on a DOPC layer depressed the current.26 Figure 3 displays current versus semi-integrated current plots derived from the data of potential-step experiments carried out in four representative systems. A best fit line is drawn through the slope of the plots at longer time scales. In all the plots a decrease in the current transient value at -0.7 V compared to the current transient value at -0.6 V is noted. The data derived from the gramicidin-modified DOPC and DOPC0.4 PS coated electrodes tend more or less to a linear plot. The slopes of the plots derived from the data from experiments at gramicidin-modified DOPC-coated electrodes in electrolyte with Dy3+ and at DOPC-0.25 B[a]p electrodes show marked increases at short time scales. The value of the intercept of the i versus m plots on the m axis (i0β-1) is variable and less than the theoretically predicted value of 1.52 µA s1/2. The presence of Dy3+ in solution decreases the value of the intercept on the i axis (i0) of the long time scale plot as well as decreasing the value of the intercept on the m axis. On the other hand, the presence of the phospholipid PS in the DOPC layer increases the value of i0 by a factor greater than 5 and increases the value of the intercept on the m axis. It is also seen that, in the plots derived from the potential step experiments carried out at the gramicidin-modified DOPC0.25 B[a]p-coated electrodes, the intercepts on the i and m axes are increased. Figure 4 shows the effect of chlorpromazine in the electrolyte solution on the differential capacity-potential

Figure 4. Differential capacity (C) versus potential (-E) plots of the DOPC-coated mercury electrodes in electrolyte 0.1 mol dm-3 KCl with 0.01 mol dm-3 buffer and 4.5 µmol dm-3 chlorpromazine in solutions of the following pH values: (s) pH 9.42 with borate buffer and (- - -) pH 5.18 with acetate buffer. Thin dashed lines refer to C versus -E plot of pure DOPC monolayer-coated electrode in the absence of chlorpromazine.

plots of a monolayer of DOPC. The capacitance-potential curves of a pure DOPC-coated electrode do not change significantly going from high to lower pH solution.17 In the presence of chlorpromazine in solution, the capacitypotential plots are modified. The values of the capacity at -0.4 V decrease in solution of high pH. In the same experiment, the capacitance peaks of the capacitancepotential plots of DOPC-coated electrodes are considerably altered to form one large peak. At solution pH’s below 7, the capacity at -0.4 V is increased. At these solution pH values, the two capacitance peaks of the capacitancepotential plots of DOPC-coated electrodes can be observed although they are considerably suppressed. Cyclic voltammograms of Tl at gramicidin-modified DOPC-coated electrodes in electrolytes with added chlorpromazine are displayed in Figure 5. The cyclic voltammograms obtained in solutions of pH 8.8 (Figure 5a) exhibit higher reduction

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Nelson

Figure 5. Cyclic voltammograms at 20 V s-1 of 4 × 10-4 Tl(I) in 0.1 mol dm-3 KCl electrolyte with 0.01 mol dm-3 buffer, 4.5 µmol dm-3 chlorpromazine, and 0.128 µmol dm-3 gramicidin at DOPC-coated electrodes: (a) pH 8.8 with borate buffer, (b) pH 7.4 with phosphate buffer, (c) pH 5.4 with acetate buffer.

and oxidation currents. At solution pH’s of 7.2 and below (Figure 5b and 5c, respectively), reduction currents are lower. Discussion 1. Model. The conclusion that radial diffusion of Tl+ to the gramicidin channels is not significant in the electrochemistry8 is connected with the observation that the Tl+ ion is reduced after passing through gramicidin channels of small dimension and proposed high density. Of equal relevance to this is the assertion8 that the gramicidin channels do not form clusters.33 The evidence that the Tl+ ion is reduced whereas the Cd2+ ion is not reduced, respectively, at a gramicidin-modified phospholipid-coated electrode8,25 shows that the monovalent ion selective gramicidin channels and not defects in the layer conduct the Tl+. In addition the fact that the Tl+ ion is not significantly reduced at phospholipid layer-coated electrodes modified with other peptides and ion specific ionophores nonselective to Tl+ 34 shows that in general the presence of peptides in the monolayer does not introduce marked defects into the layer. Previous work also showed that DOPC layers with additive compounds but without gramicidin are totally impermeable to Tl+, showing that in every gramicidin-modified layer with additive compounds it is only the gramicidin channel which conducts the Tl+.8 Cyclic voltammograms of the Tl(I)/ Tl(Hg) redox couple at gramicidin-modified pure phospholipid-coated electrodes with different concentrations of gramicidin do not show marked differences as the gramicidin concentration is increased,8 and the CV reduction current increases in a regular manner concurrent with an increase in gramicidin in the phospholipid tending to a maximum.25 This supports the contention that the gramicidin does not form significant clusters in pure DOPC layers. Figure 6 (top) summarizes as before8 the distribution of gramicidin channels in the DOPC layer consistent with the experimental evidence. In order to understand how monolayer fixed charge and the nature of the lipid environment affect the results (33) Finklea, H. O.; Snider, D. A.; Fedyk, J.; Sabatini, E.; Gafni, Y.; Rubinstein, I., Langmuir 1993, 9, 3660-3667. (34) Nelson, A. J. Chem. Soc., Faraday Trans. 1991, 87, 1851-1856.

Figure 6. (Top) Simplified model of the distribution of the gramicidin monomolecular channels in a section of phospholipid monolayer (B) sandwiched between the two conducting phases electrolyte (C) and mercury electrode (A), respectively. d denotes the average distance between the pore centres, 2r is the pore diameter, and m is the pore length (from ref 8). (Bottom) Schematic model of a potential profile across a lipid monolayer (B) adsorbed on mercury (A) to electrolyte (C) at an applied negative potential (Ea). The location of the gramicidin channel (D) in the lipid layer is shown in relation to the potential (φ1) on the monolayer surface and the potential (φ2) within the mouth of the channel. (a) Neutral lipid monolayer in nonadsorbing electrolyte; (b) neutral lipid monolayer in electrolyte with specific adsorbing cations; (c) mixed neutral and negatively charged lipid monolayer in nonadsorbing electrolyte.

obtained in this study, it is helpful to consider the charge structure of the interface. It is assumed that the PZC of the DOPC-coated mercury electrode is similar to that of an uncoated mercury electrode, which is at -0.4 V,28 and that the average capacitance of the lipid monolayer at potentials more positive than -0.8 V is 2 µF cm-2. As a result, the charge value on the electrode at the potential -0.7 V can be estimated as -0.6 µC cm-2. This small charge is compensated by the counterions, K+, in K+ electrolyte. At the same applied potential, when divalent and trivalent cations are adsorbed on the DOPC monolayer, a small concentration of the adsorbed ions will be necessary to compensate this negative charge on the electrode. As an example, a negative charge of 0.6 µC cm-2 on the electrode can be compensated by 3.1 × 10-12 mol cm-2 Mg2+ adsorbed on the lipid, which is about 1.2% of the surface concentration of lipids (∼2.56 × 10-10 mol cm-2 35). The constant for binding of Mg2+ to phosphatidylcholine is variously given but can be taken as 2 dm3 mol-1,13,14 and that for Dy3+ is of the order of 102 dm3 mol-1.13,14 Therefore in 0.05 mol dm-3 Mg2+ electrolyte (35) Moncelli, M. R.; Becucci, L.; Guidelli, R. Biophys. J. 1994, 66, 1969-1980.

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Table 1. Heterogeneous Rate Constants (kp) for the Translocation of Tl+ across the Gramicidin-Modified Monolayer and Concentrations of Tl+on the Monolayer Surface Relative to the Bulk (cm/cTl) Derived from Potential Step Experiments system: monolayer-coated electrode in electrolyte

kpa (×102 cm s-1)

cm/cTl a

DOPC in 0.1 mol dm-3 KCl at pH 7.2 DOPC in 0.1 mol dm-3 KCl with 10-3 mol dm-3 Dy3+ at pH 5.3 DOPC in 0.05 mol dm-3 Mg(NO3)2 at pH 5.3 DOPC-0.4 PS in 0.1 mol dm-3 KCl at pH 7.2 DOPC-0.25 B[a]p in 0.1 mol dm-3 KCl at pH 7.1 DOPC-0.3 retinol in 0.1 mol dm-3 KCl at pH 7.2

1.15 ((0.016)b 0.621 (0.077) 1.18 (0.138) 5.80 (0.196) 2.65 (0.370) 4.36 (0.604)

0.674 ((0.017)b 0.525 (0.070) 0.640 (0.062) 0.816 (0.005) 0.800 (0.060) 0.794 (0.039)

a From longer time scale experiment to 0.3 s at -0.6 V calculated from semi-integral analysis of current transient. Results quoted as mean and range of measurements from two consecutive transients except where indicated. b Mean and standard deviation of measurements from five consecutive transients.

and in the presence of 10-3 mol dm-3 Dy3+ in K+ electrolyte, a maximum of 9% of the DOPC on the electrode is bound to these two ions respectively. This more than compensates for the electrode charge and produces its own positive potential with respect to the electrode and solution. The incorporation of the negatively charged PS into DOPC presents a further situation in that the negative potential originating from the PS at the PS-solution interface forms part of the total applied potential profile. These three cases are analogous to the examples of the potential profiles due to no specific adsorption, and the specific adsorption of cations and anions on a negatively charged electrode surface, respectively,36 and is summarized in Figure 6 (bottom). Models of the potential profile across a lipid monolayer are complicated and still subject to discussion. There are several contributions to the monolayer potential which originate from the fixed charge on the surface,13,14,37 the dipole potential,13,14,38-41 and the orientation of the bound water molecules.13,14 Figure 6 (bottom) considers only the potentials originating from the applied potential and the Coulombic charges at the interface as an initial approximation, assuming that the lipid monolayer behaves as a low dielectric inert region and that the fixed charge is present as a layer on its surface. An applied potential falls more or less linearly along the length of the channel in the low dielectric region of the monolayer,42,43 and in the absence of a fixed charge on the monolayer surface, the major fraction of the potential drop is in this region. The presence of a fixed charge determines the steepness of this potential profile and the nature of the voltage drop between the monolayer and the solution. Capacitance measurements of the pure DOPC monolayer show that the thickness of the hydrocarbon region is of the order of 1.0-1.1 nm.6 The thickness of the polar head region of a layer of DOPC is >0.7 nm.40 A monomolecular gramicidin channel has a length of 1.3 nm,44 and thus within a DOPC monolayer it will protrude a small way out of the hydrocarbon region. This present model assumes that although there are several rate barriers for the passage of the ion through the channel,45,46 (36) Stern, O. Z. Electrochem. 1924, 30, 508-516. (37) McLaughlin, S. Annu. Rev. Biophys. Biophys. Chem. 1989, 18, 113-136. (38) Furusawa, K.; Matsumura, H. Colloids Surf., A: Physicochem. Eng. Aspects 1994, 92, 95-105. (39) Brockman, H. Chem. Phys. Lipids 1994, 73, 57-79. (40) Hauser, H.; Pascher, I.; Pearson, R. H.; Sundell, S. Biochim. Biophys. Acta 1981, 650, 21-51. (41) Seelig, J.; MacDonald, P. M.; Scherer, P. G. Biochemistry 1987, 26, 7536-7541. (42) Hall, J. E.; Mead, C. A.; Szabo, G. J. Membr. Biol. 1973, 11, 75-77. (43) Jordan, P. C.; Bacquet, R. J.; McCammon, J. A.; Tran, P. Biophys. J. 1989, 55, 1041-1052. (44) Hladky, S. B.; Haydon, D. A. Curr. Top. Membr. Transp. 1984, 21, 327-372. (45) Hille, B. Ionic Channels of Excitable Membranes; Sinauer Associates: Massachusetts, 1984; pp 291-394. (46) Jordan, P. C. J. Membr. Biol. 1984, 78, 91-102.

a significant rate-determining step for channel transport is located within the channel mouth and thus it is very sensitive to the double-layer structure and potential profile at this point. The sensitivity is manifest in the effect of the potential profile on the mean concentration of Tl+ outside the channel mouth entrance on the surface of the monolayer and also in its influence on the channel entry process itself. In the potential profile, a potential, φ1, can be identified outside the pore mouth on the monolayer surface. The effect of this potential on the concentration of Tl+ in this region (cm) is given by the Boltzmann factor:47

cm ) cTl exp(-φ1F/RT)

(8)

Within the mouth of the pore, a second potential, φ2, is located immediately beyond the free energy barrier of pore entry.48 This will influence the heterogeneous rate constant in the field direction for the passage of the ion across the channel-modified monolayer (kp), according to the following relation:6,48

kp ) k0 exp[-R(φ2 - φ1)F/RT]

(9)

where R is the transfer coefficient, which equals 0.5 when it operates over a symmetrical rate barrier. k0 is the heterogeneous rate constant for the passage of Tl+ across the channel-modified monolayer in the absence of an electric field. In Figure 6 (bottom) the location of these potentials is drawn with respect to the channel mouth, and it can be seen that both the fixed positive charge and the fixed negative charge will affect the values of φ1 and φ2 relative to those on DOPC in K+ electrolyte. 2. Chronoamperometry: Effect of Electrolytes, K+, Mg2+, and K+-Dy3+, and Monolayer Additives, PS, B[a]p, and Retinol. The decreased values of the intercept on the m axis compared to the theoretically predicted value of 1.52 µA s1/2 in the i versus m plots displayed in Figure 3 are interpreted as due to decreases in the monolayer surface concentration of Tl+ (cm) compared to the bulk (cTl). Table 1 displays values of the rate constant of translocation across the monolayer (kp) and values of cm/cTl. These are calculated from the i versus m plots derived from the various systems at the longer time scale and at the applied potential of -0.6 V. The variations of cm and kp can be explained by reference to the model in Figure 6 (bottom) and the influence of the potentials φ1 and φ2 on these parameters. On gramicidin-modified DOPC-coated electrodes in K+ electrolyte, the apparent decrease in concentration of Tl+ on the surface of the monolayer may correspond to a positive potential originating from the monolayer surface. This is consistent with previous reports which show that the effective ion concentration near a zwitterionic phospholipid layer (47) Cai, M.; Jordan, P. C. Biophys. J. 1990, 57, 883-891. (48) Kotyk, A.; Janacek, K.; Koryta, J. Biophysical Chemistry of Membrane Functions; John Wiley and Sons: Chichester, U.K., 1988; p 152.

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surface differs from that in the bulk owing to the redistribution of ions in the electrostatic, hydration, and steric-repulsion fields of the lipid layer.13 On gramicidinmodified DOPC-coated electrodes in K+ electrolyte with added Dy3+, the adsorbed Dy3+ further decreases the concentration of Tl+ on the monolayer surface as well as decreasing its translocation rate through the channel. This can be interpreted as being caused by the increased positive potentials, φ1 and (φ2 - φ1) (Figure 6 (bottom) (b)). In Mg2+ electrolyte, the influence on kp is not evident, since presumably the change in bulk electrolyte from K+ to Mg2+ affects Tl+ transport in the channel due to factors in addition to fixed charge. On gramicidin-modified DOPC-0.4 PS monolayers, the concentration of Tl+ on the monolayer surface as well as its translocation rate through the channel is increased. This can be explained as due to the negative charge of PS, which renders the potentials φ1 and (φ2 - φ1) increasingly negative compared to those on gramicidin-modified DOPC monolayers in K+ electrolyte (Figure 6 (bottom) (c)). The effect of B[a]p and retinol in increasing the parameters kp and cm/cTl associated with the translocation of Tl+ across the gramicidin-modified layer (Table 1) can be due to a number of factors, some of which alter the potential profile across the monolayer. Firstly, these additive compounds thicken the monolayer,8,17 which can stress the monolayer-channel junction such that the monolayer surface itself forms part of the mouth of the channel which is a kind of vestibule.49,50 In this case, the applied potential will fall along this vestibule section as well as the channel itself. Secondly, it has been shown that aromatic compounds shift the PZC in the negative potential direction when adsorbed on mercury due to the interaction of the π-electrons with the mercury surface.51,52 Analogous interactions between polyaromatic and polyconjugated compounds and phospholipid layers on mercury would alter the potential profile across the layer in a similar manner to that of negative fixed charge. In both situations, the changes of the potentials φ1 and (φ2 - φ1) to presumably more negative values will influence both the concentration of Tl+ on the monolayer surface and its entry rate into the gramicidin channel mouth. The observation that the Tl+ concentration (cm) prior to the channel translocation in these systems (see Table 1) is closer to the solution bulk concentration supports the contention that these compounds alter the potential profile at the lipid-water interface. The variation of the reduction current with potential (see Figure 3) can be interpreted in various ways. One possibility is that the conformation of the gramicidin in relation to the lipid alters with increasing negative potential, leading to a decrease in the reduction current. Work on this problem is continuing. The changes in the slope of the derived i versus m plots going from short to long time scales in most of the potential-step experiments deviate from the behavior expected of the ion if it conforms to a CE or an ireversible electrode process.23,24 The nonlinearity of the plot at short time scales could arise because of the difference in the surface concentration of Tl+ compared to the bulk concentration at t ) 0. This effect could be accentuated if the equilibration between cm and cTl is not so rapid as seen for example in the systems where positive fixed charge is present adsorbed on the monolayer surface and additive compounds are present which thicken the layer.8,17 There is another factor which (49) Jordan, P. C. Biophys. J. 1987, 51, 297-311. (50) Jordan, P. C. Biophys. J. 1984, 45, 1091-1100. (51) Gerovich, M. A.; Rybal’chenko, G. F. Zh. Fiz. Khim. 1958, 32, 109-115. (52) Gerovich, M. A.; Olman, O. G. Zh. Fiz. Khim. 1954, 28, 19-25.

Nelson

Figure 7. Plots of (A) kp derived from cyclic voltammograms (kp(CV)), (B) kp (kp(CA)), and (C) cm/cTl, derived from 0.3 s time scale potential (-0.6 V) step experiments, with 4 × 10-4 Tl(I) mol dm-3 in 0.1 mol dm-3 KCl electrolyte, 0.01 mol dm-3 buffer (pH < 4 glycine/HCl, pH 4-6 acetate, pH 6-8.5 phosphate, pH > 8.5 borate/NaOH), 0.128 µmol dm-3 gramicidin, and chlorpromazine at DOPC-coated electrodes. (2) no chlorpromazine; (f) 1.125 µmol dm-3 and (9) 4.5 µmol dm-3 chlorpromazine. In part A the error bars refer to the combined standard errors of the intercept and the slope of the linear regression fit (from which the rate constant is calculated) in the analysis of the CV peak currents. In parts B and C, kp(CA) and cm/cTl, respectively, are expressed as a mean derived from duplicate current transients in each system and the error bars denote the range.

might lead to the nonlinearity of the i versus m plots derived from experiments with systems of DOPC layers with the polyunsaturated additives. This is the formation of clusters of the gramicidin molecule because of its interaction with the polyunsaturated compound.53 Investigations on this problem are currently in progress. 3. Chlorpromazine Interaction with DOPC. The capacitance-potential plot of the DOPC-coated electrode in the presence of chlorpromazine (Figure 4) reflects the (53) Engelke, M.; Bojarski, P.; Diehl, H. A.; Kubicki, A. J. Membrane Biol. 1996, 153, 117-123.

Monomolecular Gramicidin Channel Function

solution properties of chlorpromazine. At solution pH values of about 10, chlorpromazine is adsorbed within the monolayer almost as a neutral molecule and the decreased capacitance minimum values indicate a thicker monolayer. At lower solution pH, interactions of the protonated chlorpromazine with the lipid layer are altered, as indicated by changes in the characteristics of the capacitance peaks in the capacitance-potential plot and increases in the capacitance minimum value. The nature of the cyclic voltammograms of the Tl(I)/ Tl(Hg) couple at the gramicidin-modified coated electrode in the presence of chlorpromazine displayed in Figure 5 shows a change in the electrode mechanism as the chlorpromazine molecule becomes charged. The derived values of kp using the Nicholson and Shain analysis are displayed in Figure 7A. These exhibit high values at high solution pH and low values at low solution pH. The extent of these effects is correlated with the concentration of chlorpromazine in solution. Figures 7B and C display values of kp and cm/cTl, respectively, derived from the potential step experiments at -0.6 V and at the longer time scale. The trends in the variation of kp with solution pH in Figure 7B are similar to those displayed in Figure 7A. The higher values of kp observed in Figure 7A are due to the fact that the derivation of kp from the CV measurement includes results from short time scale measurements, where deviations from the proposed models exist. The values of kp and cm/cTl at high solution pH are similar to those arising from the effect of unsaturated compounds in the DOPC monolayer, and at low solution pH they are similar to those arising from the effect of fixed positive charge in the DOPC monolayer (cf. Table 1). The kp and cm/cTl values support the results from the CV and the capacitance measurements in reflecting the changing interaction of chlorpromazine with the monolayer with change in solution pH. Figure 7B and C shows that the crossover in the effect of chlorpromazine in the monolayer on the translocation of Tl+ in the channel occurs around solution pH 7.2, which is lower than its pKa value in solution by about two units.

Langmuir, Vol. 13, No. 21, 1997 5651

Conclusions Negatively charged phosphatidylserine (PS) when incorporated into DOPC layers at 0.4 mole fraction increases the gramicidin-mediated permeability through the layers by increasing the concentration of Tl+ on the monolayer surface and increasing the rate of translocation of the ion in the channel by a factor of about 5.0. Positively charged ions which adsorb onto lipid layers have the opposite effect; for example, 10-3 mol dm-3 Dy 3+ added to K+ electrolyte decreases the Tl+ concentration on the monolayer surface and decreases the rate of translocation of Tl+ in the channel by a factor of about 1.9. Polyunsaturated compounds (e.g. all-trans-retinol) when incorporated into DOPC monolayers increase the Tl+ concentration on the monolayer surface and increase the translocation rate of Tl+ in the channel by a factor of about 3.8 at longer experimental time scales. These findings are commensurate with a model where a significant rate-determining step for channel translocation is the entry of the ion into the channel mouth. This is very sensitive to the charge structure and consequent potential profile at the lipidwater interface, properties which are affected by the presence of additive compounds in the monolayer. The chlorpromazine interaction with DOPC layers depends on its charge. At high solution pH its effect on the gramicidin-mediated permeability is similar to that of a polyunsaturated compound whereas at low solution pH its positive charge dominates its influence on the transport of Tl+ in the channel. The crossover in the effects of chlorpromazine on the gramicidin channel translocation of Tl+ occurs around solution pH 7.2, which is about two units below its pKa value. Acknowledgment. This work was suppported by the Ministry of Defence (U.K.) grant “Phospholipid layers” D/ACSA(R)/3/10/4/3/20. Thanks are also extended to Dr. Herman van Leeuwen for reading the manuscript and to Dr. Dan Bizzotto for discussions on this subject. LA970030O