11302
J. Phys. Chem. 1995,99, 11302-11305
Rapid Transport of Protons across Membranes by Aliphatic Amines and Acids Arvind Srivastava, Shanteri Singh, and G. Krishnamoorthy* Chemical Physics Group, Tata Institute of Fundamental Research, Homi Bhabha Road, Bombay 400 005, India Received: February IO, 1995; In Final Form: April 21, 1995@
The pH gradient generated across membranes can be dissipated efficiently by means of a single-turnover cycle of aliphatic amines and acids present in the membrane. The kinetics of this single-turnover process was in the submillisecond time scale and was measured by creating pH gradients (ApH) using a temperature jump. The observed ApH decay process was identified as due to rapid transmembrane movement of the neutral form of either the acid or the amine. The rate constant was estimated to be in the range (1 -6) x lo3 s-I in the case of aliphatic amines.
Introduction Facilitated transport by carriers is one of the mechanisms by which ions and small molecules are translocated across biological membranes.' Although there are a variety of mechanisms by which carriers function,* they are common in the following aspect: transport across membrane is achieved by multiple tumover of the carrier similar to catalytic cycles (Figure 1A). The rate of transport or the flux is often dictated by the rate of transmembrane movement of either the carrier-ion complex or the bare ~ a r r i e r .Identification ~ of the rate-determining step is achieved either by kinetic analysis of transport3 or by a suitable perturbation of the flux! In contrast to the multiple turnover process (Figure lA), one could envisage a transport process which would involve mainly a single tumover of the carrier. Such a situation could arise from very slow rate of transmembrane movement of either the loaded or the free carrier (Figure lB,C). Localized charge on either the free or the loaded carrier could be the cause of its reduced ~ermeability.~ (In the case of protonophores such as CCCP, SF6847, etc., the charge is highly delocalized on the entire molecules making them highly membrane-permeable.) Thus when the time scale of observation is small when compared to the time scale associated with the translocation of the charged form of the carrier, a multiple-tumover carrier becomes, phenomenologically, a single-turnover carrier. Although every carrier could be described in this way, a clear experimental separation of the single-tumover process from the multipletumover process requires the kinetics of transmembrane movement of the charged form to be substantially slower than that of the uncharged form. However observations on longer time scales will be dominated by the slow multiple-turnover process of the carrier in such cases also. One of the advantages offered by such carriers is the ability to study the kinetics of transmembrane translocation with ease by observations in short time scales. Proton transport by aliphatic amines and acids is expected to offer the possibility of experimentally resolving the single- and multiple-tumover processes. This is due to the presence of localized charge in one of the protonation states (ionized carboxyl or protonated amine) and thereby drastically reducing their permeability. Free fatty acids and polyamines are endogenous components in many tissues and cells6 and hence proton transport by these agents is physiologically relevant. Uncoupling of oxidative phosphorylation in mitochondria by fatty @
Abstract published in Advance ACS Abstracts, July 1, 1995.
0022-365419512099-11302$09.OOl0
acids7 and amines8 have been reported. Dissipation of transmembrane proton gradient is the most accepted mechanism of uncoupling by fatty acids. Fatty acids are also involved in the signal transductiongand in the control of ion channel opening.I0 As mentioned above, transport of protons by acids and amines could be achived in two ways: (i) a very rapid movement of the neutral carrier resulting in translocation of protons in a stoichiometric manner (Figure 1B,C) and (ii) slow cycling process (Figure 1A) resulting in translocation of protons in a catalytic manner. Although dissipation of a proton gradient is expected to occur largely by the latter process?-* the former process could be relevant in signal transduction and control of ion-channel opening. Apart from providing information on the kinetics of these processes, a detailed study on the mechanism of action of aliphatic amines and acids will offer direct insight into the kinetics of transmembrane movement of small neutral molecules. In this work, we have studied the kinetics of singletumover proton transport by aliphatic acids and amines using the method based on temperature-jump developed in our laboratory." This technique allows us to monitor the kinetics of transmembrane movement of molecules in submillisecond time scales.
Experimental Section Soybean phospholipid vesicles were made and characterized as described earlier." CCCP, SF6847, nigericin, and the longchain amines and acids were obtained from Sigma Chemical co. The partitioning of the aliphatic amines and acids into the lipid vesicles was estimated as follows: The vesicle suspension having either the amine or the acid was filtered through an ultrafiltration membrane (Amicon, YM10). The initial filtrates were assayed for either the amine or the acid. The amines were assayed by the formation of Schiff s base (absorption at 420 nm) with salicylaldehyde. n-Octanoic acid was assayed by titrating with NaOH using phenolphthalein as indicator. Control experiments were run in the absence of vesicles in the buffer in order to get the partition coefficient. Typically, the percentage of n-octylamine and n-octanoic acid in the membrane phase was about -25% and -15% at a lipid concentration of 1.5 mg mL.-' T-jump experiments were performed as described earier." Typically, the samples had sonicated soybean lipid vesicles at a concentration of 1.5 mg mL-' suspended in 150 mM KCl and 30 mM Tris at pH 7.5. The medium inside the vesicle was 150 mM KC1, 30 mM phosphate, 5 mM pyranine at pH 0 1995 American Chemical Society
Letters
J. Phys. Chem., Vol. 99, No. 29, 1995 11303
A
C
6
I ti+-
I-i
* +H
PH
PH
IN
OUT
MEMBRANE
Figure 1. Schemes for the transport of protons by multiple tumover process (A) and single tumover processes in the cases of long-chain amines (B) and acids (C). In the case of the multiple tumover process (A), both the forms (P-and PH) are membrane permeable. In contrast, in the case of single-turnover processes (B and C), the charged forms have very low permeability across the membrane.
TABLE 1: Rate of Relaxation of ApH across Vesicular Membrane Aided by Either n-Octylamine or n-Octanoic Acid under Various Conditions no.
experimental system
kaDD0(103 S-I)
1 2
1.5 mM n-octylamine -do10 pM valinomycin -do- f 20 pM tetraphenyl boron -do- f 2 mM chlorodecane 1.5 mM n-octanoic acid -do10 pM valinomycin -do2 mM chlorodecane
9.5 9.1 9.1 9.5 15.7 16.7 15.7
3 TIME
Figure 2. Typical traces showing the relaxation of ApH (seen as decrease in pyranine fluorescence intensity) by the multiple-turnover process in the cases of monensin and the single-tumover processes in the cases of n-octylamine (A) and n-octanoic acid (B). The concentration of various carriers and the time per division are as follows: trace a, 0.25 mM n-octylamine and 1.2 ms; trace b, 0.5 mM n-octylamine and 360 ps; trace c, 15 pM monensin and 24 ms; trace d, 1 mM n-octanoic acid and 158 ps; trace e, 3.8 mM n-octanoic acid and 45 ps; trace f, 5 mM n-octanoic acid and 45 ps. Arrow indicates the time of application of T-jump.
7.5. Application of 2'-jump (-3.0 "C) caused a ApH of about 0.10 unit in 2.5 ps." The decay of ApH was monitored by the fluorescent pH indicator pyranine." The ApH decay curves were analyzed as described earlier.4$'' The intemal buffering power (B) of the vesicles was estimated as described earlier.''
Results and Discussion Figure 2 shows typical traces of relaxation of ApH by either n-octylamine or n-octanoic acid. Two points are worthy of observation: (i) the time scale associated with the transport process is quite fast (< 1 ms) and (ii) the amplitude of the relaxation (fluorescence change) increased with an increase in the concentrations of the carrier. The latter observation could be due to incomplete dissipation of ApH at lower concentrations of the carrier. One of the reliable identification of a single tumover process is the strict stoichiometry between the number of ions/ molecules transported and the number of the transporter molecule. This could lead to incomplete dissipation of ApH. The incomplete dissipation of ApH observed in the cases of n-octylamine and n-octanoic acid contrasts with those usually observed with catalytic multiple tumover processes such as normally observed in the cases of ionophores and protonophores" (Figure 2). In the latter cases, the entire gradient was dissipated by multiple turnover of the carrier leading to constant (and a higher level of) amplitude of ApH relaxation traces at all concentrations of the carrier.
4 5
6 7
+ + +
kappwas estimated from the time constant t of the experimental traces of ApH relaxation (kapp= 6-l). Each measurement is an average of 8-10 experiments. The error in measurement is -10%.
The observed relaxation traces (Figure 2) could be fitted to a single-exponentialfunction.I2 The apparent rates (kapp= l/z, where z is the exponential time constant) obtained from such traces are given in Table 1. The values of kappare in the range (1-5) 104 ~ 1 . The transporf process could be visualized as follows: we start with the initial state in which the carrier is distributed equally between the two sides of the membrane (Figure lB,C). Aliphatic acids and amines are expected to orient with their functional groups close to the membrane-water interface and their aliphatic chain extending into the hydrophobic core. Application of a 2'-jump creates a ApH ( p b u t < pHin) in -3 pus." The ApH is the result of the larger decrease in the pK of tris[(hydroxymethyl)amino]methane (Tris) buffer in which the vesicles are suspended when compared to the decrease in the pK of phosphate buffer trapped inside the vesicle.I1 The larger decrease in p b U tis rapidly followed by a shift in the protonation equilibrium of the amine toward RNH3+ (RCOOH in the case of acids). We assume that the protonation equilibrium on either side of the membrane is faster when compared to the time scale of our observation. The larger shift in the protonation equilibrium of the carrier at the outer half of the membrane leads to a concentration gradient of the neutral form ([RNH2]0 < [RNH2li or [RCOOH], > [RCOOH],]). Since the neutral forms are expected to have high mobility: it will redistribute across the membrane in order to neutralize the concentrationgradient. The resulting decreases in [RNH2], will shift the protonation equilibrium at the inner half of the membrane. This would result in a stoichiometric increase in the concentration of H+ in the inner aqueous phase which is felt and reported by the intemal pH indicator, pyranine. Thus the influx of protons occurs in an indirect way. Description of this process as "single-turnover transport of protons" may sound inappropriate in the case of
11304 J. Phys. Chem., Vol. 99, No. 29, 1995
Letters of the carrier:
where the subscripts o and i refer to the outer and inner half of the membrane, respectively (Figure 2B), and the superscript f refers to the final state (the state at the end of the observed relaxation process, Figure 2). This behavior can be quantitatively accounted for (in the case of amine, for example) as shown below. The distribution of the amine in the initial state (before the T-jump) can be described by the following equations:
0.8 2
t J a
5
0.6
Z
P la X a
0.4
J
w
a 0.2
0
0
1.0
2.0
3.0
(3)
[n-OCTYLAMINE] , m M
Figure 3. Increase in the amplitude of n-octylamine-aided relaxation of ApH on the total concentration of n-occtylamine. The dotted line shows the amplitude corresponding to complete decay of ApH as observed in a multiple turnover process in the case of monensin. The smooth line is the simulation (using eqs 7-11) with the buffering capacity (B) of 28 nmoUpWmg of lipid and pK of amine in the membrane as 8.3.
amines since no dissociable proton is carried by the amine during the movement of the neutral form (Figure 1B). However, this process is associated with protonation on one side of the membrane and deprotonation on the other side resulting in a situation which is very similar to transport of protons across the membrane. As mentioned earliar, the transport process observed in fast time scales (Figure 2) does not have any significant contribution from the cycling multiple-turnover process due to its slow nature. Observations at longer time scales (such as seconds to minutes) will reveal the multiple turnover process as observed in earlier studies.6-s The rapid transport process (Figure 2) would lead to buildup of a concentration gradient of the ionic form (RNH3+ or RCOO-) across the membrane. The low permeability of the ionized form would be the reason for their accumulation and consequently the slow kinetics of multiple turnover. Any transmembrane movement of the charged carrier (or carrierion complex) would have resulted in a diffusion potential which would oppose the net transport of protons, unless relieved by a counterion flux such as valinomycin-aided K+ flux, for example.4 The absence of any significant effect on the observed rates by (i) the presence of valinomycin in a K+ medium, (ii) the increases of membrane dielectric constant by the addition of chlorodecane, l 3 and (iii) the presence of tetraphenyl boron anion (TPB-) which would have increased the permeability of RNH34+ (Table 1) shows the noninvolvement of the charged carriers in the observed rapid transport process. It is important to realize that ApH cannot be entirely neutralized by the single-turnover process. The inability to neutralize the ApH completely in a single-turnover process was demonstrated by the data shown in Figure 3. The amplitude of the relaxation process (which is proportional to the extent of ApH neutralized) did increase with the increases in the concentration of the transporter but saturated at a level significantly lower than that obtained in the case of multipleturnover processes such as that observed in the case of nigericin. The magnitude of the residual ApH which remains unneutralized would be equal to the gradient of the ionized form
[RNH,];
+ [RNH3+]i + [RNH,]; + [RNH3+]: = R,
(4)
where pK refers to the pK of the amine in the membrane and the superscript i refers to the initial state. R, is the total concentration of amine in the membrane phase and was determined by assaying, for amine, the initial filtrate of the vesicle suspension passed through an ultra filtration membrane. Since pHf = pH,', we also have [RNH,11 = [RNHzI'o
(5)
[RNH3+]i = [RNH3+]a
(6)
The final state at the end of the observed relaxation process (Figure 2) can be described by the following equations:
(7)
The end of the relaxation process is related to the achievement of the condition given in eq 10. It should be noted that due to low permeability of RNH3+, the condition shown in eq 6 is not applicable in the final state. We also have the relationship governing the internal buffering power, B: [RNH3+1: - [RN&+]f PH: - PHf
=B
(11)
In our experimental conditions, pH:, pHf, Rt, B, and p q are experimentally determined quantities. The five unknowns [RNHJf,, [RNH3+]z, [RNH,];, [RNH3+]f, and pHf could be determined by solving eq 7- 11. Graphical solution of these nonlinear equations by assuming various values of pK' gives pHf. pHf is related to the amplitude of the observed relaxation
Letters process (Figure 2) as amplitude a (pH: - pH:). The dependence of pHf on Rr, the total concentration of amine, and comparison with the experimentally obtained dependence (Figure 3) has given us the value of pK of amine in the membrane as 8.3. This simulation also indicates that ApH cannot be entirely dissipated by a single-turnover cycle of the amine. A similar analysis could be applied in the case of fatty acids also. The rate constant (ka) associated with the transmembrane movement of RNH2 and RCOOH (Figure 1B,C) was calculated in the following way. We start with the initial conditions mentioned earlier. Immediately following the T-jump (prior to the relaxation process) a concentration gradient of the neutral form of the carrier (RNH;, or RCOOH) is set up across the membrane([RNHp]o < [RNH2],; [RCOOH], > [RCOOH],). The magnitude of this gradient can be calculated from the known value of ApKIAT for the intemal and extemal buffers used (phosphate and Tris, respectively).l' The initial slope of the relaxation process gives the initial rate of the change of pH,. By using the internal buffer capacity, B (see Experimental Section), this rate could be converted into the rate of proton influx, J:
The value of ka calculated in this way is -2 x lo3 s-l in the case of n-octylamine. This value is comparable to the value of rate constant associated with the movement of neutral carriers across the m e m b r a ~ ~ e . Further, ~.'~ the present estimation of k, could be considered as more direct when compared to analyses of the multiple-tumover p r o c e ~ s e s . ~ The . ' ~ observation of a single-tumover process, in the present situation, has helped in dissecting the transport cycle into elementary steps. Table 2 gives the values of k, for a series of alkylamines. Since the membrane-water partition constant depends on the chain length, the value of k, were calculated by taking this into account. It can be seen that ka decreases with the increase in the chain length. This could be due to hindrance offered by the long chain in the flip-flop motion of RNHp across the membrane.
J. Phys. Chem., Vol. 99, No. 29, 1995 11305 TABLE 2: Rate Constant (k,) Associated with the Transmembrane Movement of Alkylamines ~
no.
carrier
k (lo3s-')
1 2
n-butylamine (C4) n-octylamine (CS) CSamine C I amine ~ C12 amine
5.8 2.2 1.3 1.1 1.o
3 4
5
Thus our experiments and analysis have enabled, for the first time, the estimation of the rate of transmembrane movement of amines and fatty acids which have a variety of physiological functions including signal t r a n ~ d u c t i o n . ~The ~ ' ~submillisecond kinetics associated with the transmembrane movement would be compatible with their role in signal transduction processes.
References and Notes (1) (a) Heytler, P. G. Methods Enzymol. 1979,55,462. (b) Pressman, B. C. Annu. Rev. Biochem. 1976, 45, 501. (2) (a) Krishnamoorthy, G.; Ahmed, I. In Biomembranes Structure and Function-The State of the Art; Gaber, B. P., Easwaran, K. R. K., Eds.; Adenine Press: 1992; p 95. (b) Terada, H. Biochim. Biophys. Acta 1981, 639, 225. (3) (a) Kasianowicz, J.; Benz, R.; McLaughlin, S. J. Membr. B i d 1987, 95, 73. (b) Benz, R.; McLaughlin, S. Biophys. J. 1983, 41, 381. (4) Ahmed, I.; Krishnamoorthy, G. Biochim. Biophys. Acta 1990,1024, 298. ( 5 ) Parsegian, A. Nature 1969, 221, 844. (6) (a) Hamilton, James, A.; Civelek, Vildan, N.; Kamp, Frits.; Tomheim, Keith.; Corkey, Barbara E. J . B i d . Chem. 1994, 269, 20852. (b) Kamp, F.; Hamilton, J. A. Biochemistry 1993, 32, 11074. (c) Janne, J.; Alhonen, L.; Leinonen, P. Ann. Med. 1991, 23, 241. (d) Gutknecht, J. J . Membr. Bioi. 1988, 106, 83. (7) (a) Wojtczak, L.; Schonfeld, P. Biochim. Biophys. Acta 1993,1183, 41. (b) Schonfeld, P.; Schild, L.; Kunz, W. Biochim. Biophys. Acta 1989, 977, 266. (c) Kohnke, D.; Ludwig, B.; Kadenbach, B. FEBS Lett. 1993, 336, 90. (8) Garlid, K. D.; Nakashima, R. A. J. Bioi. Chem. 1983, 258, 7974. (9) Nishizuka, Y. Science 1992, 258, 607. (IO) (a) Ordway, R. W.; Walsh, J. V. Jr.; Singer, J. J. Science 1989, 244, 1176. (b) Huang, J. M.-C.; Xian, H.; Bacaner, M. Proc. Natl. Acad. Sci. U S A . 1992, 89, 6452. (1 1) Krishnamoorthy, G. Biochemistry 1986, 25, 6666. (12) Grzesiek, S.; Dencher, N. A. Biophys. J . 1986, 50, 265. (13) Dilger, J. P.; McLaughlin, S. G. A,; McIntosh, T. J.; Simon, S. A. Science (Washington, D.C.) 1979, 206, 1196. (14) Prabhananda, B. S.; Ugrankar, M. M. Biochim. Biophys. Acta 1991, 1070, 481.
JP950385L
