J. Phys. Chem. 1994,98, ll00I-ll003 11001

Permselectivity of Voltage-Gated Alamethicin Ion Channel Studied by Microamperometry

Tomokazu Matsue,' Hitoshi Shiku, Hirnshi Yamada, and Isamu Uchida Department of Molecular Chemistry and Engineering. Faculty of Engineering, Tohoku University, Sendai 980-77, Japan

Received: May 4, 1994; In Final Form: July 21, 1 9 9 P

The permeability of R u ( N H ~ ) ~ ~ + , and I- through voltage-gated alamethicin ion channels in a planar lipid membrane was investigated by amperometric measurements using a microelectrode located near the membrane. The redox current at the microelectrode initiated by application of a potential pulse to the BLM was quantitatively analyzed to obtain permselectivity of the channel. The permeability of Fe(CN)& was ca. I O times smaller than that of Ru(NH3).P due to electrostatic repulsion, indicating that the channel gives a high barrier to the permeation of multicharged negative ions.

Introduction

Microelectrcdes are being widely used for obtaining informa- tion on biological processes occurring in localized regions, particularly extra-!-) and intracellular fluids?,s Recently, microelectrochemical measurements have been refined and systematized as scanning electrochemical microscopy (SECM)"' to view localized electrochemical properties of biological surfaces?-1° In the present paper, we used SECM technology to investigate redox ion transport through alamethicin ion channels in a bilayer lipid membrane (BLM). Alamethicin. a polypeptide antibiotic, aggregates and forms voltage-gated ion channels in artificial and biological membranes." Unlike biological channel proteins in plasma membranes. alamethicin ion channels were thought to he only weakly ion selective." Despite numerous studies of alamethicin ion channels, the mechanism of channel formation and thus the origin of ion selectivity are still in dispute.13 We determined the permeability of multicharged redox ions, R u ( N H ~ ) ~ ~ + and Fe(CN)63-. through alamethicin ion channel (Figure I ) and found that the channel strongly prefers Ru(NH&?+ to Fe(CN)$. Since the micro- electrode responds only to the redox species permeated through the ion channels, the present procedure is more straightforward to determine the flux of redox ions, as compared with the methods based on measurements of membrane potential or membrane current which is influenced by all the ionic species dissolved in the so lu t i~n . '~ J~

Experimental Section

The microelectrode was fabricated by sealing a Fl microwire (radius 7.5 pm) to soft glass tubing.!" The tip was then polished successively with 1.0.3, and 0.05 pm alumina powder to obtain a disk-shaped electrode, which was then followed by rinsed with distilled water under supersonication. The radius tip including insulating sheath was ca. 50 pm. BLMs were formed in a pinhole (radius 0.3 nun) of a Teflon sheet from a 2 mg/mL solution of soybean lecithin (Wako Chemicals, Osaka) in decane under a microscope." The formation of a BLM was confirmed by the disappearance of the fringe and by capacitance (0.5 pF/ an2). Total ionic (or membrane) currents and redox currents were monitored by using a self-made bipotentiostat connected with the microdisk electrode (Wl), a Ag/AgCI electrode (W2). a Ag/AgCI (saturated KCI) electrode (RE), and a PI wire (CE) (see Figure 1). Micromovement of the microdisk electrode was

Absmct published in Advonce ACS Abstracts, October I , 1994.

0022-3654/94/2098- I1001$04.50/0

I I BP h I

Cis BLM Tmfl.5

Figure 1. Simultaneous measuremenls of ionic and redox current: BP = bipotentiostate: WI = Pt microdisk electrode (Pt radius. 7.5 pm); W2 = AglAgCl e l d e ; RE = Ag/AgCI electrode connectul to virmal ground: CE = pt wire.

performed by means of a motor-driven X-Y stage (PMC200P. New Port). Unless otherwise stated, the electrolytes on either side of the BLM were 1.0 M KCI. In this work, we denote the compartment connected to virtual ground to (RE) as the trans side. Alamethicin was added to the electrolyte solution so that the concentrations in the both c o m p m e n t s were equal. Alamethicin channels opened when the membrane potential exceeded 50 mV, and the total ionic current increased expo- nentially with the membrane potential. We measured the dependence of the total ionic current on the alamethicin concentration. At membrane potentials of 60-90 mV, the total ionic current increased with the 56th power of alamethicin concentration in the range 1 x 10-8-2.4 x IO-' g/mL, indicating that the average ion channel consisted of 5.6 alamethicin molecules. Therefore, the average inner radius of the channel is ca. 0.7 nm.Is Ru(NH3)6(CIO& (synthesized according to the literatu&" or K3Fe(CN)6 (GR grade, Kanto Chemicals, Tokyo) was added to the cis compartment, and the redox ion permeation through the ion channels was detected at the microelectrode located in the trans compartment (Figure 1). Since the concentrations of KCI in trans and cis compart- ments are equal and in large excess over that of the redox ion, the diffusion potential induced by the difference in redox ion concentration is virtually negligible. The theoretical responses at the microelectrode were calculated by the digital simulation" incorporating the DuFort-Frankel algorithm.18 The flux of

0 1994 American Chemical Society

11002 J. Phys. Chem., Vol. 98, No. 43, I994 Letters

TABLE 1: Permselectivity of Alamethicin Ion Channel Embedded in a BLM

ion PR D x 105/cm2 s-l Stokes radius/nmb

200

% . CI 100 E f 0

0

0 1 2 3 llme / sec

Figure 2. Responses of ionic current and reduction current for Ru- (NH3)63+. (A) Potential pulse applied to the BLM. (B) Total ionic current. (C) Reduction current at the microdisk electrode at -0.25 V vs Ag/AgCl. Distance: a, 11 pm; b, 10 pm; c, 27 pm. (0) Simulated based onfdx = 1.2 x mol/(cm2 s). Concentration of Ru(NH~)~~+ in the cis side is 8.4 mM.

redox ion permeated through the voltage-gated ion channels was determined by curve-fitting the experimental and simulated responses. The distance between the microelectrode tip and the BLM was determined from a working curve showing the oxidation current for 1.0 mM We(CN)a (GR grade, Kanto Chemicals, Tokyo) in the trans compartment vs distance profile.lob All the measurements were carried out at 25 OC.

Results and Discussion

The application of the membrane potential allows alamethicin to form channels in the BLM and therefore allows redox ion in the cis side to permeate through the channels into the trans side. The redox ions thus permeate to the trans side and diffuse into the electrode surface to give reduction current responses. Figure 2 shows typical responses of the total ionic current and the reduction current of Ru(NH3)63+ upon application of a potential pulse of a 1.0 s period. The ionic current responds quickly to the membrane potential change, indicating that the formation and disruption of ion channels in the BLM induced by the membrane potential are very rapid. Since the concentration of KCl was in great excess over that of RI I (NH~)~~+ , the ionic current was mainly determined by the movements of K+ and C1-. The total ionic flux (ftod) through the BLM calculated from the maximum ionic current was found to be 8.2 x equiv/(cmzs). On the other hand, the response of the reduction current for R u ( N H ~ ) ~ ~ + at the microdisk electrode was slow because of the diffusion effect. The BLM without ion channels exhibits a high barrier to transmembrane movement of Ru- (NH3)63f, as is evidenced by observation of no reduction current before the potential pulse application.

The magnitude and shape of the response at the microelec- trode depend on the distance between the electrode tip and the BLM surface. The response becomes rapid and large as the distance decreases. The circles shown in Figure 2 are the

K+ 1 .o 2.w (1.0) 0.11 c1- 0.5 2.0' (1.0) 0.11 RU(NH3)63f 0.27 f 0.05 0.6W (0.29) 0.37 Fe(CN)63- 0.03 f 0.01 0.76' (0.38) 0.29

1- 0.3 f 0.2 2.0' (1.0) 0.11 a Diffusion coefficient in solution. Numbers in parentheses are the

relative values. Determined by the Stokes-Einstein equation.20 Ref- erence 22. Determined from cyclic voltammetry. e Reference 23. fDetermined by analyzing membrane potentials.'l It was difficult to determine accurate values because of the large membrane resistance.

theoretical responses obtained from the digital simulation. Reasonable agreement between the experimental and simulated curves was found when the flux of R u ( N H ~ ) ~ ~ + through the BLM was assumed to be 1.2 x mol/(cm2 s). The ion movement through the alamethicin channel is primarily due to migration and diffusion. From the Nernst-Planck equation,Ig one can find that migration induced by a large electric field is the major factor for the movement of the redox ions. Indeed, despite the formation of ion channels in the BLM, no response of the reduction current for RU(NH3)a3+ was observed when negative potential pulses were applied to the BLM.

The permselectivity of the alamethicin ion channel (PR, relative to the permeability of K+) can be estimated from the flux of total ions and the flux of redox ions v;Cdox). The potentiometric measurements revealed that the PR value of C1- was 0.50.1z On the basis of these results, we used the following equation to determine the PR values for redox ions through the alamethicin ion channels;

Fe(CN)63-f <0.1

where C represents the concentration of the indicated species and z is the charge on the redox ion. From eq 1, the PR value for Ru(NH&~+ was calculated to be 0.27. When the pulse height increased from 70 to 90 mV, PR remained the same value althoughf,,,d increased from 4 x to 1.4 x equiv/ (cm2 s). The PR values of F ~ ( C N ) G ~ - and I- were also determined from the redox current responses at the microelec- trode (Figure 3). In these cases, we applied negative potential pulses to the BLMs. The PR value for Fe(CN)63- determined by the microamperometric measurement agrees with that obtained by the usual method based on analyzing membrane potentials.12 It should be noted that the PR value for Fe(CN)63- is extremely small compared to that for R u ( N H ~ ) ~ ~ + . The permselectivity of the alamethicin ion channel can be discussed based on the diffusion coefficient of these ions in solution.

The PR value of R u ( N H ~ ) ~ ~ + is comparable to the relative diffusion coefficient, indicating that the selectivity reflects the difference in mobilities of K+ and Ru(NH3),j3+. The large discrepancy in the values of PR and relative diffusion coefficient for Fe(CN)63- is probably caused by electrostatic repulsion. Although the inner radius of the average alamethicin ion channel (0.7 nm) is considerably large compared to the Stokes radius of Fe(CN)63-, the carbonyl groups located at the inner wall of the alamethicin ion channelsz0 repulse the multicharged ion. Alamethicin channels have been thought to be weakly cation selective.l2 The present results demonstrate that the channel gives a high barrier to the permeation of multicharged negative ions.

The responses of the oxidation current for I- observed at the microelectrode also agree well with those from digital simulation when the distance is relatively large. However, the deviation

Letters J. Phys. Chem., Vol. 98, No. 43, 1994 11003

(2) Wightman, R. M.; May, L. J. ; Michael, A. C. Anal. Chem. 1988, 60, 769A-779A.

(3) (a) Ciolkowski, E. L.; Cooper, B. R.; Jaukowski, J. A.; Jorgenson, J. W.; Wightman, R. M. J . Am. Chem. SOC. 1992, 114, 2815-2821. (b) Tanaka, K.; Kobayashi, F.; Isogai, Y.; Iizuka, T. Bioelectrochem. Bioenerg. 1991, 26, 413-421.

(4) (a) Chien, J. B.; Wallingford, R. A,; Ewing, A. G. J . Neurochem. 1990, 54, 633-638. (b) Lau, Y. Y.; Abe, T.; Ewing, A. G. Anal. Chem. 1992,64, 1702-1705. (c) Chen, T. K.; Lau, Y. Y.; Wong, D. K. Y.; Ewing, A. G. Anal. Chem. 1992,64, 1264-1268. (d) Abe, T.; Lau, Y. Y.; Ewing, A. G. Anal. Chem. 1992.64, 2160-2163.

( 5 ) (a) Uchida, I.; Abe, T.; Itabashi, T.; Matsue, T. Chem. Lett. 1990, 1227-1230. (b) Matsue, T.; Koike, S.; Abe, T.; Itabashi, T.; Uchida, I. Biochim. Biophys. Acta 1992, 1101, 69-72. (c) Matsue, T.; Koike, S.; Uchida, I. Biochem. Biophys. Res. Commun. 1993, 197, 1283-1287.

(6) (a) Engstrom, R. C.; Weber, M.; Wunder, D. J.; Burgess, R.; Winquist, S. Anal. Chem. 1986,58,844-848. (b) Engstrom, R. C.; Meancy, T.; Tople, R.; Wightman, R. M. Anal. Chem. 1987, 59, 2005-2010. (c) Engstrom, R. C.; Wightman, R. M.; Kristensen, E. W. Anal. Chem. 1988, 60, 652-656. (d) Engstrom, R. C.; Pharr, C. M. Appl. Chem. 1989, 61, 1099A-1104A.

(7) Kwak, J.; Bard, A. J. Anal. Chem. 1989, 61, 1221-1227. (8) (a) Lee, C.; Wipf, D. 0.; Bard, A. J.; Bartels, K.; Bovik, A. C.

Anal. Chem. 1991, 63, 2442-2447. (b) Bard, A. J.; Fan, F. F.; Pierce, D. T.; Unwin, P. R.; Wipf, D. 0.; Zhou, F. Science 1991, 254, 68-74. (c) Pierce, D. T.; Unwin, P. R.; Bard, A. J. Anal. Chem. 1992, 64, 1795- 1804. (d) Lee, C.; Kwak, J.; Bard, A. J. Proc. Natl. Acud. Sci. 1990, 87, 1740-1743. (e) Pierce, D. T.; Bard, A. I. Anal. Chem. 1993, 65, 3598- 3604.

(9) (a) Scott, E. R.; White, H. S. J . Membr. Sci. 1991,58, 71-87. (b) Scott, E. R.; White, H. S.; Phipps, J. B. Anal. Chem. 1993, 65, 1537- 1545.

(10) (a) Yamada, H.; Matsue, T.; Uchida, I. Biochem. Biophys. Res. Commun. 1991,180, 1330-1334. (b) Yamada, H.; Shiku, H.; Matsue, T.; Uchida, I. Bioelectrochem. Bioenerg. 1994, 33, 91 -93.

(11) (a) Mueller, P.; Rudin, D. 0. Nature 1968, 217, 713-719. (b) Boheim, G. J . Membr. Biol. 1974, 19, 277-303. (c) Eisenberg, M.; Hall, J. E.; Mead, C. A. J . Membr. Biol. 1973, 14, 143-176.

(12) Menestrina, G.; Voges, K. P.; Jung, G.; Boheim, G. J . Membr. Biol. 1986, 93, 111-132.

(13) (a) Rizzo, V.; Stankowski, S.; Schwartz, G. Biochemistry 1987, 26, 2751-2759. (b) Archer, S. J.; Ellena, J. F.; Cafko, D. S. Biophys. J. 1991, 60, 389-398.

(14) Yamada, H.; Shiku, H.; Matsue, T.; Uchida, I. J. Phys. Chem. 1993, 97, 9547-9549.

(15) Hanke, W.; Boheim, G. Biochim. Biophys. Acta 1980, 596,456- 462.

(16) Fergusson, J. E.; Love, J . L. Inorganic Chemistry; McGraw-Hill: New York, 1972; Vol. 13, pp 208-213.

(17) The determination of flux at the electrode surface was basically same as those used by Kwak and Bard (ref 7) except the initial and boundary conditions as follows: For t = 0; 0 5 r, 0 5 z 5 d, CO = 0: For 0 < tp (pulse period); 0 5 r 5 a, z = 0, CO = 0; a < r, z = 0, Xdaz = 0; 0 5 r, z = d, DaCdaz = f&x (constant): For tp < t; 0 5 r 5 a, z = 0, CO = 0; a < r, z = 0, Kdaz = 0; 0 5 r, z = d, Xdaz = 0.

(18) Feldberg, S. W. J . Electroanal. Chem. 1990, 290, 49-65. (19) Bard, A. J.; Faulkner, L. R. Electrochemical Methods; John

(20) Fox, R. O., Jr.; Richards, F. M. Nature 1982, 300, 325-330. (21) (a) Krysinski, P.; Tien, H. T. Bioelectrochem. Bioenerg. 1986, 16,

185-191. (b) Bender, C. J.; Tien, H. T. Anal. Chim. Acta 1987, 201. (22) Atkins, P. W. Physical Chemistry, 2nd ed. (in Japanese); Tokyo

Kagaku, Dojin: Tokyo, Chapter 26. (23) Sawyer, D. T.; Boberts, J. L., Jr. Experimental Electrochemistry

for Chemists; John Wiley: New York, p 77.

Wiley: New York, 1980; Chapter 1.

U ~ ~ 1 0 - ~ -

t p) 1x10-4-

t 0-

a 0

> .- c m - I

0 1 2 3 Time I sec

Figure 3. Responses of redox current for Fe(CN)63- (A) and I- (B) at the microdisk electrode. The currents were normalized based on the total ionic current. (A) Concentration of Fe(CN)63- in the cis side is 0.10 M. Membrane potential pulse: height 70 mV, period 1 s. Electrode potential: 0.00 V vs AglAgCl. Distance: a, 13 pm; b, 27 pm. (0) Simulated based onfEdox = 1.0 x mol/(cm2 s). (B) Concentration of I- in the cis side is 25 mM. Concentration of KC1 in the trans and cis sides is 0.10 M. Membrane potential pulse: height 90 mV, period 1 s. Electrode potential: 0.50 V vs Ag/AgCl. Distance: a, 10 pm; b, 28 pm. (0) Simulated based onfiedox = 2.0 x

mol/(cm2 s).

of the response from the simulated curve becomes eminent as the electrode tip approaches the BLM surface (Figure 3). The electrooxidation of I- forms 13-, which diffuses back to the BLM surface and deposits 12 into the BLM. The I2 in the BLM acts as a carrier to transport I- from trans to cis side. Tien and co-workers21 found that 12 in BLMs is an effective mediator for permeation of I-. This 12-assisted permeation enhances the flux of I- and brings about increases in the oxidation current for I- compared to that expected from the permeation through the ion channels. Although the accurate PR value for I- is difficult to obtain, the PR value seems to be close to the value of c1-.

These results establish a basis of quantitative understanding of permeability of natural and artificial voltage-gated ion channels toward redox ions. Since a single alamethicin channel of average size has a conductance of ca. 4 nS,I5 the channel density in the present BLM is ca. 0.2 channeVpm*. It is difficult to quantify the number of channels sampled in the measure- ments. However, by considering the diffusion length during the potential pulse, one can qualitatively say that the response reflects the permeation in the circular membrane area with radius of ca. 50 pm, where approximately 400 channels were located. The use of a smaller electrode will enable single channel recording of redox ion permeation.

References and Notes (1) Adams, R. N. Anal. Chem. 1976, 48, 1126A-1138A.