| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
* Institut für Biophysik, Johannes Kepler Universität-Linz, Linz, Austria; and A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia
Correspondence: Address reprint requests and inquiries to Peter Pohl, Tel.: 43-732-2468-9269; Fax: 43-732-2468-9270; E-mail: peter.pohl{at}jku.at.
 |
ABSTRACT |
|---|
 TOP ABSTRACT REFERENCES |
|---|
Weak acids and bases constitute a class of pharmacologically important substances. Since most of them have an intracellular target, investigation of their membrane transport has attracted much attention in the past. At physiological pH most weak acids are anions, A–, while their neutral form, AH, is much more membrane-permeable. Consequently, proton-transfer reactions in the immediate membrane vicinity play an important role in the permeation process (1
,2). They include several steps: 1), the diffusion of A– to the membrane; 2), proton uptake; 3), permeation of AH across the membrane; 4), dissociation; and 5), diffusion of A– into the bulk.
Belonging to the class of weak acids as well, protonophores (uncouplers of oxidative phosphorylation) permeate membranes by essentially the same mechanism (3). Kinetic modeling always revealed that the rate constant for the backward movement of the anionic form is several orders-of-magnitude smaller than the rate constant of the neutral form. For example, the respective numbers for carbonyl cyanide m-chlorophenylhydrazone amount to 175 s–1 and 12,000 s–1 in chlorodecane-containing membranes (4). Without the increase of membrane dielectric permittivity, 
m, mediated by the polar solvent, the difference in the permeabilities would have been even larger. In the case of fatty acids the difference rate constants of AH and A– is extremely large. They translocate through lipid bilayers exclusively in their protonated form because of the low anion permeability (5,6).
The difference in the transport rates for AH and A– has been attributed to the energetic barrier by which any membrane opposes the movement of charged entities. In addition to the neutral energy term that is equal for AH and A–, the interaction of A– with lipid bilayers is characterized by the electrical Born, image, and dipole contributions (7).
The Born energy barrier is a function of ion radius, a, and ion charge, q,
 | (1) |
w is the dielectric constant of water. Thus, the partition of the salicylicate anion with a = 1.25 nm (8) into the membrane costs 6.7 kcal mol–1. Since the partition coefficients are by far the major determinants of the relative magnitudes of the permeability coefficients (9), the ratio, r, of AH and A– permeabilities, Pm,AH and Pm,A–, can be estimated using the Boltzmann distribution:
 | (2) |
In contrast to the prediction made by Eq. 2, Thomae et al. (10) claimed r = 66 for salicylic acid (SA). Theoretically, the divergence may be attributed to image and dipole contributions, which are ignored in Eq. 2. However, for an uncharged lipophilic substance, the reported Pm,AH of 3.5 x 10–7 cm/s is unreasonably low. It violates the Overton rule. The SA octanol/water partition coefficient of 300 (10) suggests Pm,AH 
1 cm/s. This is in line with Pm,AH = 0.7 cm/s determined earlier (11).
Strikingly enough, the ratio of the two experimental Pm,AH values (0.7: 3.5 x 10–7) is close to r from Eq. 2. Even more surprising is that the Pm,AH reported by Thomae et al. (10) corresponds to the membrane permeability one would expect for a charged species according to the Overton rule. A possible explanation for this coincidence is offered by the fact that Thomae et al. (10) have monitored SA transport into buffer-free vesicles. The accompanying proton transport is likely to lower intravesicular pH. In turn, the resulting pH gradient opposes acid diffusion. This phenomenon is very well known and the reverse effect has widely been used to load vesicles (12,13).
Exceptions to the Overton rule are rare (14). They are mostly due to proteinaceous transporters (15) or membrane phase transitions (16). To clarify whether Thomae et al. (10) have discovered a new exception or whether Overton still rules, we simultaneously monitored AH and A– permeations across solvent-free planar lipid bilayers (17) by electrochemical microscopy and current measurements under voltage-clamp conditions. The former method makes use of scanning pH-sensitive microelectrodes (18,19). Small changes in proton concentration close to the membrane surface are detected that accompany weak acid transport. As expected, the pH shift adjacent to the membrane depended on bulk pH (Fig. 1). Calculation of hydrogen ions flux, jp, were performed according to the equation (20,21)
 | (3) |
H, OH, and b denoted the aqueous diffusion coefficients and individual unstirred fluid layer thicknesses of H+, OH–, and buffer, respectively. The value b was the buffer capacity of aqueous solutions. At pH 7, the contribution of the two first terms can be neglected. The value jp amounts to 2 x 10–11 mol cm–2 s–1 at 2 mM SA.
|
 | (4) |
Pm,A– is very low, as revealed by the lack of an increment in electrical conductivity after addition of salicylic acid at pH 7 (Fig. 2). The pKa = 2.75 indicates that at pH = 3, the transmembrane concentration gradient of A– exceeds that of AH nearly twofold. Consequently, Eq. 2 predicts a jAH/jA– ratio of 36,500. Based on jp = 10–9 mol cm–2 s–1 measured at 2 mM SA, jm,A– = 2.8 x 10–14 mol cm–2 s–1 is expected. From the experimentally observed increase in current, I, an anion flux, jm,A–, of
 | (5) |
), whereas the protonated forms are transported at rates between 0.1 s–1 (23) and 15 s–1 (24). The increase in the salicylate-mediated conductivity at low pH is consistent with previous data (25).
|
,19),
 | (6) |
 | (7) |
 | (8) |
 | (9) |
At the membrane-water interface, the fluxes of all species are required to be equal to zero, except for J2, so that
 | (10) |
The numerical solutions are derived, assuming that the rates of chemical reactions (like dissociation/recombination of water, buffer, and SA) are very high compared to the rate of diffusion through the unstirred fluid layer, so that the local chemical equilibrium is maintained (18,19). The best agreement between model and experiment was achieved for Pm,AH = 1.2 cm s–1 (Fig. 1). It should be noted that setting Pm,AH to 3.5 x 10–7 cm/s as reported by Thomae et al. (10) resulted in the lack of a pH shift in the membrane vicinity. The discrepancy with the experiment indicated that the result of Thomae et al. is not correct.
Thus, salicylate transport across membranes is well described by the pH-partition mechanism assuming that only the protonated species diffuses through the membrane. At physiological pH, lipid bilayer permeation of the acid anion is negligible. The result agrees well with negligible partitioning of the charged species into n-octanol. Overton still rules. The energetic barrier imposed by partitioning of a charged species into the membrane still holds—in contrast to the report by Thomae et al. (10).
Submitted on March 1, 2006; accepted for publication April 3, 2006.
|
REFERENCES |
|---|
|
TOPABSTRACTREFERENCES |
|---|
2. Walter, A., and J. Gutknecht. 1984. Monocarboxylic acid permeation through lipid bilayer membranes. J. Membr. Biol. 77:255–264.[CrossRef][Medline]
3. McLaughlin, S., and J. P. Dilger. 1980. Transport of protons across membranes by weak acids. Physiol. Rev. 60:825–863.
4. Kasianowicz, J., R. Benz, and S. McLaughlin. 1984. The kinetic mechanism by which CCCP (carbonyl cyanide m-chlorophenylhydrazone) transports protons across membranes. J. Membr. Biol. 82:179–190.[CrossRef][Medline]
5. Kamp, F., and J. A. Hamilton. 1992. pH gradients across phospholipid membranes caused by fast flip-flop of unionized fatty acids. Proc. Natl. Acad. Sci. USA. 89:11367–11370.
6. Pohl, E. E., U. Peterson, J. Sun, and P. Pohl. 2000. Changes of intrinsic membrane potentials induced by flip-flop of long-chain fatty acids. Biochemistry. 39:1834–1839.[CrossRef][Medline]
7. Flewelling, R. F., and W. L. Hubbell. 1986. The membrane dipole potential in a total potential model. Applications to hydrophobic ion interactions with membranes. Biophys. J. 49:541–552.
8. Zhou, Y., and R. M. Raphael. 2005. Effect of salicylate on the elasticity, bending stiffness, and strength of SOPC membranes. Biophys. J. 89:1789–1801.
9. Orbach, E., and A. Finkelstein. 1980. The nonelectrolyte permeability of planar lipid bilayer membranes. J. Gen. Physiol. 75:427–436.
10. Thomae, A. V., H. Wunderli-Allenspach, and S. D. Kramer. 2005. Permeation of aromatic carboxylic acids across lipid bilayers: the pH-partition hypothesis revisited. Biophys. J. 89:1802–1811.
11. Gutknecht, J., and D. C. Tosteson. 1973. Diffusion of weak acids across lipid bilayer membranes: effects of chemical reactions in the unstirred layers. Science. 182:1258–1261.
12. Madden, T. D., P. R. Harrigan, L. C. Tai, M. B. Bally, L. D. Mayer, T. E. Redelmeier, H. C. Loughrey, C. P. Tilcock, L. W. Reinish, and P. R. Cullis. 1990. The accumulation of drugs within large unilamellar vesicles exhibiting a proton gradient: a survey. Chem. Phys. Lipids. 53:37–46.[CrossRef][Medline]
13. Harrigan, P. R., K. F. Wong, T. E. Redelmeier, J. J. Wheeler, and P. R. Cullis. 1993. Accumulation of doxorubicin and other lipophilic amines into large unilamellar vesicles in response to transmembrane pH gradients. Biochim. Biophys. Acta. 1149:329–338.[Medline]
14. Cantor, R. S. 2001. Breaking the Meyer-Overton rule: predicted effects of varying stiffness and interfacial activity on the intrinsic potency of anesthetics. Biophys. J. 80:2284–2297.
15. Al-Awqati, Q. 1999. One hundred years of membrane permeability: does Overton still rule? Nat. Cell Biol. 1:E201–E202.[CrossRef][Medline]
16. Blume, A. 1986. Lipid phase transitions: water and proton permeability. Methods Enzymol. 127:480–486.[Medline]
17. Krylov, A. V., P. Pohl, M. L. Zeidel, and W. G. Hill. 2001. Water permeability of asymmetric planar lipid bilayers: leaflets of different composition offer independent and additive resistances to permeation. J. Gen. Physiol. 118:333–339.
18. Antonenko, Y. N., G. A. Denisov, and P. Pohl. 1993. Weak acid transport across bilayer lipid membrane in the presence of buffers—theoretical and experimental pH profiles in the unstirred layers. Biophys. J. 64:1701–1710.
19. Antonenko, Y. N., P. Pohl, and G. A. Denisov. 1997. Permeation of ammonia across bilayer lipid membranes studied by ammonium ion selective microelectrodes. Biophys. J. 72:2187–2195.
20. Borisova, M. P., L. N. Ermishkin, E. A. Liberman, A. Y. Silberstein, and E. M. Trofimov. 1974. Mechanism of conductivity of bimolecular lipid membranes in the presence of tetrachloro-trifluoromethyl-benzimidazole. J. Membr. Biol. 18:243–261.[CrossRef][Medline]
21. Pohl, P., S. M. Saparov, and Y. N. Antonenko. 1998. The size of the unstirred layer as a function of the solute diffusion coefficient. Biophys. J. 75:1403–1409.
22. Brunaldi, K., M. A. Miranda, F. Abdulkader, R. Curi, and J. Procopio. 2005. Fatty acid flip-flop and proton transport determined by short-circuit current in planar bilayers. J. Lipid Res. 46:245–251.
23. Kleinfeld, A. M. 2000. Lipid phase fatty acid flip-flop: is it fast enough for cellular transport? J. Membr. Biol. 175:79–86.[CrossRef][Medline]
24. Kamp, F., D. Zakim, F. L. Zhang, N. Noy, and J. A. Hamilton. 1995. Fatty acid flip-flop in phospholipid bilayers is extremely fast. Biochemistry. 34:11928–11937.[CrossRef][Medline]
25. Gutknecht, J. 1990. Salicylates and proton transport through lipid bilayer membranes: a model for salicylate-induced uncoupling and swelling in mitochondria. J. Membr. Biol. 115:253–260.[CrossRef][Medline]
This article has been cited by other articles:
 |
|
 |
|
  A. Missner, P. Kugler, S. M. Saparov, K. Sommer, J. C. Mathai, M. L. Zeidel, and P. Pohl Carbon Dioxide Transport through Membranes J. Biol. Chem., September 12, 2008; 283(37): 25340 - 25347. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
