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Selective Na+ Transport through Phospholipid Bilayer Membrane by a Synthetic Calix[4]arene Carrier Takashi Jin,*,† Masataka Kinjo,† Tomiyasu Koyama,† Yoko Kobayashi,‡,§ and Hajime Hirata‡,| Section of Intelligent Materials and Device, Research Institute for Electronic Science, Hokkaido University, Sapporo 060, Japan, and Department of Biochemistry, Jichi Medical School, Minami-Kawachi-machi, Kawachi-gun, Tochigi 329-04, Japan Received August 8, 1995. In Final Form: March 4, 1996X Alkali cation transport by tetrakis(ethoxycarbonylmethyl ether) 1 and tetrakis(methyl ether) 2 of p-tertbutylcalix[4]arene through soybean phospholipid bilayer membranes has been investigated by measurements of electric currents and dynamic 23Na NMR spectra. When the calix[4]arene ether 1 was incorporated into a planar phospholipid bilayer membrane which separated two chambers filled with 100 mM NaCl solutions, electric currents resulting from sodium ion fluxes across the planar bilayer membrane were generated upon applying external voltages between two chambers. On the other hand, incorporation of the methyl ether 2 into the planar bilayer did not generate electric currents resulting from sodium ion fluxes under the same experimental condition with 1. The current-voltage (I-V) relationships in XCl/XCl (X ) Li, Na, K, Rb, Cs) systems showed that the calix[4]arene 1 selectively transported sodium ions through the phospholipid bilayer membrane. From the measurement of reversal potentials in XCl/NaCl systems, it was found that the permeability of sodium ions was much larger than that of other alkali cations, by a factor of 17 (for K+) or higher (for Li+, Rb+, Cs+). Incorporation of 1 into large unilamellar vesicles (LUVs) in NaCl aqueous solution gave rise to dynamic 23Na NMR spectra arising from 1-mediated Na+ exchange across the LUV bilayer membrane. The rates (1/τNa+in) of Na+ transport by 1 were ca. 4-fold smaller than that by a naturally occurring ionophore, monensin. The kinetics for 1-mediated Na+ transport follows a facilitated diffusion model in which one calix[4]arene molecule complexes with one sodium ion at the water-membrane interface and sodium ions are transported to the opposite interface by the diffusion of cationic Na+-1 complexes.
Introduction In the past 2 decades, many studies of metal ion transport by synthetic ionophores such as crown ethers and cryptands have been conducted with bulk liquid membranes and supported liquid membranes.1 However, there are a limited number of reports of synthetic ionophore-mediated ion transport through phospholipid bilayer membranes as models for biological membranes.2 It is well-known that naturally occurring antibiotic ionophores such as monensin and valinomycin mediate alkali cation transport through biological membranes.3 On the other hand, the ionophoric activities of synthetic †
Hokkaido University. Jichi Medical School. Present address: Gunma University. | Present address: Himeji Institute of Technology. X Abstract published in Advance ACS Abstracts, April 15, 1996. ‡ §
(1) For example, see: (a) Maruyama, K.; Tsukube, H.; Areko, T. J. Am. Chem. Soc. 1982, 104, 5197. (b) Shinkai, S.; Kinda, H.; Araragi, Y.; Manabe, O. Bull. Chem. Soc. Jpn. 1983, 56, 559. (c) Izatt, R. M.; Lamb, J. D.; Hawkins, R. T.; Brown, P. R.; Izatt, S. R.; Christensen, J. J. J. Am. Chem. Soc. 1983, 105, 1782. (d) Izatt, R. M.; Dearden, D. V.; Brown, P. R.; Bradshaw, J. S.; Lamb, J. D.; Christensen, J. J. J. Am. Chem. Soc. 1983, 105, 1785. (e) Lamb, J. D.; Brown, P. R.; Christensen, J. J.; Bradshaw, J. S.; Garrick, D. G.; Izatt, R. M. J. Membr. Sci. 1983, 13, 89. (f) Behr, J. P.; Kirch, M.; Lehn, J. M. J. Am. Chem. Soc. 1985, 107, 241. (g) Kobuke, Y.; Tabushi, I.; Oh, K.; Aoki, T. J. Org. Chem. 1988, 53, 5933. (h) Wienk, M. M.; Stolwijk, T. B.; Sudho¨lter, E. J. R.; Reinhoult, D. N. J. Am. Chem. Soc. 1990, 112, 797. (2) (a) Thomas, C.; Sautery, C.; Castaing, M.; Gray-Bobo, C. M.; Lehn, J. M.; Plumere, P. Biochem. Biophys. Res. Commun. 1983, 116, 981. (b) Castaing, M.; Morel, F.; Lehn, J. M. J. Membr. Biol. 1986, 89, 251. (c) Shinar, H.; Navon, G. J. Am. Chem. Soc. 1986, 108, 5005. (d) Castaing, M.; Lehn, J. M. J. Membr. Biol. 1987, 97, 79. (e) Nakano, A.; Xie, Q.; Mallen, J. V.; Echgoyen, L.; Gokel, G. W. J. Am. Chem. Soc. 1990, 112, 1287. (f) Kobuke, Y.; Ueda, K.; Sokabe, M. J. Am. Chem. Soc. 1992, 114, 7618. (g) Fyles, T. M.; James, T. D.; Kaye, K. C. J. Am. Chem. Soc. 1993, 115, 12315. (h) Xie, Q.; Li, Y.; Gokel, G. W.; Herna´ndez, J.; Echegoyen, L. J. Am. Chem. Soc. 1994, 116, 690. (i) Murillo, O.; Watanabe, S.; Nakano, A.; Gokel, G. W. J. Am. Chem. Soc. 1995, 117, 7665.
S0743-7463(95)00674-3 CCC: $12.00
compounds in phospholipid bilayer membranes are known to a lesser extent. Our interest is to determine whether synthetic ionophores have the capacity for selective ion transport across phospholipid bilayer membranes in a similar manner as antibiotic ionophores. Phospholipid bilayers are very thin and molecular-size membranes (d ≈ 30 Å) in comparison with liquid membranes and supported liquid membranes. To form stable bilayer membranes, the ionophore incorporated into the bilayer must possess a high lipophilicity. So far lipophilic compounds derived from crown ethers and cryptands have been used in the study of synthetic ionophore-mediated ion transport through phospholipid bilayer membranes. For example, in the middle of 1980s, Lehn and his coworkers2a,b,d studied Na+ and K+ transport by cryptands with pendant alkyl chains in large unilamellar vesicle (LUV) systems using pH measurements. More recently, Xie et al.2h studied Na+ transport by a series of 12 monoazacrown ethers containing amide groups in LUV systems using dynamic 23Na NMR technique. In this work, we chose ionophoric calix[4]arenes 1 and 2 to test their abilities to selectively transport sodium ions in planar bilayer membrane systems (Chart 1). Calixarenes are a new class of host molecules prepared by phenol-formaldehyde condensation.4 In contrast to the macrocyclic polyether compounds such as crown ethers, calixarenes possess high lipophilicities because they are made from benzene units. In addition, the ether derivatives of calix[4]arenes exhibit high Na+ selec(3) For example, see: (a) Mueller, P.; Rudin, D. O. Biochem. Biophys. Res. Commun. 1967, 26, 398. (b) Henderson, P. J. F.; McGivan, J. D.; Chanppell, J. B. Biochem. J. 1969, 111, 521. (c) Sandeaux, R.; Sandeaux, J.; Gavach, C.; Brun, B. Biochim. Biophys. Acta 1982, 684, 127. (4) For example, see: (a) Gutsche, C. D.; Muthukrishnan, R. J. J. Org. Chem. 1978, 43, 4905. (b) Gutsche, C. D.; Levin, J. A. J. Am. Chem. Soc. 1982, 104, 2652. (c) Gutsche, C. D. Prog. Macrocycl. Chem. 1987, 3, 93.
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Figure 1. A schematic diagram of a planar bilayer system. Chart 1. Structures of Ether Derivatives of p-tert-Butylcalix[4]arene 1 and 2
branes and solid-supported liquid membranes.5,7,9,13-17 Herein we wish to report that the calix[4]arene ether 1 mediates selective Na+ transport through phospholipid bilayer membranes. On the basis of the kinetic data obtained from dynamic 23Na NMR spectra with a chemical shift reagent,18 we also describe a facilitated diffusion model for the 1-mediated Na+ transport. To our knowledge, this is the first report of selective Na+ transport by a synthetic compound, calix[4]arene ether 1, through phospholipid bilayer membranes. Experimental Section
tivities5-12 in ion extraction from a water phase to an organic phase and/or in ion transport through dichloromethane liquid membranes. Interestingly, these compounds have amphiphilic structures composed of a hydrophilic ion binding cavity and benzene moieties which make them soluble in lipid membranes. Therefore, it seemed to us that the ether derivatives of calix[4]arene could be easily incorporated into phospholipid bilayer membranes where they should exhibit selective Na+ transport abilities. So far ion transport properties of calixarene ether derivatives have been studied using bulk liquid mem(5) Alfieri, C.; Dradi, E.; Pochini, A.; Ungaro, R.; Andreetti, G. D. J. Chem. Soc., Chem. Commun. 1983, 1075. (6) McKervey, M. A.; Seward, E. M.; Ferguson, G.; Ruhl, B. L.; Harris, S. J. J. Chem. Soc., Chem. Commun. 1985, 388. (7) Chang, S.-K.; Cho, I. J. Chem. Soc., Perkin Trans 1 1986, 211. (8) Arduini, A.; Pochini, A.; Reverberi, S.; Ungaro, R.; Andreetti, G. D.; Ugozzoli, F. Tetrahedron 1986, 42, 2089. (9) Arunand-Neu, F.; Collins, E. M.; Deasy, M.; Ferguson, G.; Harris, S. J.; Kaitner, B.; Lough, A. J.; McKervey, M. A.; Marques, E.; Ruhl, B. L.; Schwing-Weill, M. J.; Seward, E. M. J. Am. Chem. Soc. 1989, 111, 8681. (10) Arimura, T.; Kubota, M.; Matsuda, T.; Manabe, O.; Shinkai, S. Bull. Chem. Soc. Jpn. 1989, 62, 1674. (11) Ghidini, E.; Ugozzoli, F.; Ungaro, R.; Harkema, S.; El-Fadl, A. A.; Reinhouldt, D. N. J. Am. Chem. Soc. 1990, 112, 6979. (12) (a) Jin, T.; Ichikawa, K. J. Phys. Chem. 1991, 95, 2601. (b) Jin, T.; Ichikawa, K.; Koyama, T. J. Chem. Soc., Chem. Commun. 1992, 499.
Materials. Tetrakis(ethoxycarbonylmethyl ether) 1 and tetrakis(methyl ether) 2 of p-tert-butylcalix[4]arene were prepared according to the literature method.6,7 Monensin was purchased as its sodium salt from Wako Pure Chemicals (Tokyo, Japan) and recrystallized from ethanol. Phospholipids used for formation of planar bilayer membrane and large unilamellar vesicles were prepared from soybean phospholipids (Nakarai Tesque, Kyoto, Japan) and purified by the literature method.19 Analytical reagent grades of LiCl, NaCl, KCl, RbCl, and CsCl were purchased from Wako and used without further purification. Planar Bilayer Membrane System. A simplified diagram of a planar bilayer system is shown in Figure 1. Planar bilayer membranes were formed at an aperture (0.2 mm diameter) in a Teflon film (12.5 µm thick) which separated two Teflon chambers (internal volume of each chamber is 1.5 mL with surface area of 1 cm2). The side to which compounds were added was defined as “cis” chamber and the opposite side was “trans” chamber. An amplifier (CEZ-2300; Nihon Kohden, Ltd., Tokyo, Japan) was used in a voltage clamp mode to amplify the currents and to control the voltages across the bilayer membranes. The command voltage was fed to the trans chamber via an Ag/AgCl electrode (13) Izatt, R. M.; Lamb, J. D.; Hawkins, R. T.; Brown, P. R.; Izatt, S. R.; Christensen, J. J. J. Am. Chem. Soc. 1983, 105, 1783. (14) Izatt, S. R.; Hawkins, R. T.; Christensen, J. J.; Izatt, R. M. J. Am. Chem. Soc. 1985, 107, 63. (15) Goldman, H.; Vogt, W.; Paulus, E.; Bo¨hmer, V. J. Am. Chem. Soc. 1988, 110, 6811. (16) Shinkai, S.; Shiramama, Y.; Satoh, H.; Manabe, O.; Arimura, T.; Fujimoto, K.; Matsuda, T. J. Chem. Soc., Perkin Trans. 2 1989, 1167. (17) (a) Nijenhuis, W. F.; Buitenhuis, E. G.; de Jong, F.; Sudho¨lter, E. J. R.; Reinhoult, D. N. J. Am. Chem. Soc. 1991, 113, 7963. (b) Casnati, A.; Pochini, A.; Ungaro, R.; Ugozzoli, F.; Arnaud, F.; Fanni, S.; Schwing, M.-J.; Egberink, R. J. M.; de Jong, F.; Reinhoult, D. N. J. Am. Chem. Soc. 1995, 117, 2767. (18) (a) Gupta, R. K.; Gupta, P. J. Magn. Reson. 1982, 47, 344. (b) Balshi, J. A.; Cirillo, V. P.; Springer, C. S. Biophys. J. 1982, 38, 323. (c) Boulanger, Y.; Vinaly, P.; Desroches, M. Biophys. J. 1985, 47, 553. (19) Hirata, H.; Ohno, K.; Sone, N.; Kagawa, Y.; Hamamoto, T. J. Biol. Chem. 1986, 261, 9839.
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through an agar bridge and the cis chamber was grounded via an Ag/AgCl electrode through an agar bridge. The voltage was referenced to the cis side with respect to the trans side. The output signal from the amplifier was filtered at 3 kHz and then distributed to a monitoring oscilloscope or a chart recorder. All measurements were performed at 25 °C. Formation of Planar Bilayer Membranes. Planar bilayer membranes were prepared by the folding method.20 To both chambers, 0.5 mL of the aqueous solution of 100 mM alkali chloride salts containing 25 mM Hepes (N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid)-Tris (tris(hydroxymethyl)aminomethane) buffer (at pH 7.0) was injected through two syringes so that the water surface was just below the aperture. Then a 15 µL aliquot of asolectin or asolectin + calix[4]arene esters dissolved in hexane (10 mg/mL) was placed on the surface of the solutions in both chambers. After a while (ca. 10 min), hexane was vaporized leaving a phospholipid monolayer (or phospholipid monolayer containing calix[4]arene esters) at the air-water interface in both chambers. The bilayer membrane was then formed by raising the water level sequentially in both chambers above the aperture. Bilayer membranes whose electric resistance and membrane capacitance were more than 200 GΩ and 0.5-0.7 µF/cm2 were used. Incorporation of Calix[4]arene Ethers into Planar Bilayer Membranes. In order to incorporate the calix[4]arene ethers into planar bilayer membranes, two methods were utilized. One method was the “incubation” technique: after the formation of a planar bilayer membrane, small aliquots of DMSO calix[4]arene solutions were added to the cis chamber with gentle stirring. Another one was the “direct” method: a planar bilayer membrane containing calix[4]arenes was formed from the asolectin-calix[4]arene hexane solution. By using both methods, similar current levels upon the amounts of calix[4]arenes 1 and 2 were observed within experimental error. In the case of the measurement of I-V curves in XCl (cis)/ NaCl (trans) systems, the same planar bilayer membrane prepared from asolectin-hexane solution containing 1 (w/w asolectin:1 ) 500:1) was used in order to obtain reversal potentials. First, a planar bilayer membrane which separated two chambers filled with 100 mM NaCl solutions was prepared from the asolectin-hexane solution containing 1. Then the NaCl solution in the cis chamber was successively replaced with other 100 mM XCl aqueous solutions. This operation was carefully carried out to avoid the break of the planar bilayer membrane. Preparation of Large Unilamellar Vesicles. Large unilamellar vesicles (LUV) were prepared from asolectin by the freezing-thawing-sonication method.21 A 100 mg portion of asolectin was dispersed to 10 mL of 100 mM NaCl aqueous solution. The sample suspension containing asolectin was sonicated to clarify in a tip-type sonicator (Branson Model 185 Sonifier) under nitrogen atmosphere at 4 °C. Then the sonicated samples were frozen at -196 °C by gently shaking in liquid nitrogen and left to thaw at 25 °C for 15 min. The thawed milky solution was sonicated in a bath-type sonicator (Branson Model S2200) for 30 s at 25 °C. This freezing-thawing-sonication cycle was repeated 3 times. The milky suspension (2.5 mL) containing LUVs was centrifuged to separate into a water phase and vesicles pellets. The water phase was decanted and carefully replaced with 0.5 mL of 10 mM Na 5PPPi (sodium tripolyphosphate)/50 mM NaCl aqueous solution. This procedure was repeated 5 times to complete replacement of the water phase with the salt solution. A 1 mL portion of the LUV solution was taken and sufficient 1 M solution (approximately 5 µL) of DyCl3 was added to create a 10-15 ppm shift difference between intraand extravesicular sodium signals. 23Na NMR Measurements. 23Na NMR measurements were carried out on a Varian XL-200 spectrometer at 52.3 MHz. Fieldfrequency lock was used by an external D2O in the inner compartment of a coaxial 5 mm tube. The calix[4]arene ethers and monensin were prepared as 1 mM DMSO solutions and added directly to the NMR tube containing the LUVs by a microliter syringe. The solution was then allowed to equilibrate for 30 min before data acquisition. The spectra were obtained after the (20) (a) Takagi, M.; Azuma, K.; Kushimoto, U. Annu. Rep. Bio. Works Fac. Sci. Osaka Univ. 1965, 13, 107. (b) Montal, M.; Mueller, P. Proc. Natl. Acad. Sci. U.S.A. 1972, 69, 3561. (21) Pick, U. Arch. Biochem. Biophys. 1981, 212, 186.
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Figure 2. Electric currents at 100 mV in Na+/Na+ system versus amounts of the calix[4]arene 1 (b) and 2 (0) added to the cis chamber. Both chambers were filled with 100 mM NaCl solutions containing 25 mM Hepes-Tris buffer (pH ) 7.0). Microliter aliquots of DMSO solutions (100 µM) of the calix[4]arenes were added to the cis chamber under gentle stirring. The measurements of electric currents were carried out 6 times. Error bars represent a standard deviation. accumulation of 2000-5000 FIDs to improve the signal to noise ratio. All measurements were performed at 25 °C.
Results Electric Current Measurements. Since the calix[4]arene ethers 1 and 2 are neutral ionophores having no ionizable groups, it was expected that the calix[4]arenes would transport alkali cations with cationic complexes in a similar manner as valinomycin.3a,b On the basis of this expectation, we attempted to measure electric currents arising from the cation fluxes mediated by 1 and 2 through a planar bilayer membrane. Figure 2 shows the generation of electric currents (at 100 mV in NaCl/NaCl system) upon the amounts of 1 and 2 added to the cis chamber. When a microliter aliquot of the DMSO solution (100 µM) of 1 was added to the cis chamber under stirring, membrane conductance immediately increased. The value of electric current increased with increasing the amounts of 1 added to the cis chamber. In contrast to the case of 1, the addition of 2 did not increase membrane conductance under the same experimental condition with 1. Control experiments were performed where only neat DMSO was added to the cis chamber. The addition of 200 µL of DMSO did not increase membrane conductance: a control level of the current was 0.3 pA at 100 mV. These results indicate that the calix[4]arene 1 mediates ion (sodium cation or chlorine anion) fluxes across the planar bilayer membrane. For the calix[4]arene 2, we could not find ion transporting activity from the measurement of electric current using the planar bilayer system. To identify the ionic species transported by 1, we examined I-V relationships in five ionic conditions, where both chambers were filled with the same solutions of 100 mM XCl (X ) Li, Na, K, Cs, Rb). Figure 3 shows I-V curves obtained for the five ionic systems. In the NaCl/ NaCl system, electric currents were generated to afford a symmetrical I-V curve. On the other hand, in four XCl/XCl (X ) Li, K, Rb, Cs) systems, effective currents over the control level of current were not generated: only a small electric current (
