Fatty Acid Fueled Transmembrane Chloride Transport

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Cite This: J. Am. Chem. Soc. 2019, 141, 10654−10660

Fatty Acid Fueled Transmembrane Chloride Transport Ethan N. W. Howe and Philip A. Gale* School of Chemistry, The University of Sydney, Sydney NSW 2006, Australia

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S Supporting Information *

ABSTRACT: Generation of chemical gradients across biological membranes of cellular compartments is a hallmark of all living systems. Here we report a proofof-concept prototype transmembrane pumping system in liposomes. The pump uses fatty acid to fuel chloride transport, thus generating a transmembrane chloride gradient. Addition of fatty acid to phospholipid vesicles generates a transmembrane pH gradient (pHin < pHout), and this electrochemical H+ potential is harnessed by an anionophore to drive chloride efflux via H+/Cl− cotransport. Further addition of fatty acid efficiently fuels the system to continuously drive chloride transport against the concentration gradient, up to [Cl−]in 65 mM | [Cl−]out 100 mM, and is 1400 times more efficient than using an external fuel. Based on our findings from dissecting the H+/Cl− flux process with the use of different liposomal fluorescence assays, and supported by additional liposome-based 13C NMR and DLS studies; we proposed that the presence of an anionophore can induce asymmetric distribution of fatty acid, and contribute to another Cl− flux mechanism in this system.

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neutral H+/Cl− cotransport,9 as well as anion antiport processes such as Cl−/NO3− and Cl−/HCO3− exchange.10 We have previously shown that compounds 1−3 are excellent chloride anionophores 11 and can also mediate H+ /Cl− cotransport. Anionophores 1 and 3 induce H+ flux via deprotonation of the acidic NH,12 similar to the H+ transport mechanism of weak acid protonophores,13 e.g., carbonyl cyanide m-chlorophenyl hydrazone (CCCP), whereas 2 transports H+ by a coupling mechanism in accelerating the FA flip-flop process.14 The EC50 values (i.e., effective concentration of anionophore required to achieve 50% H+/ Cl− cotransport at 200 s, obtained from Hill analysis15) shown in Figure 1c were obtained from a routine liposomal HPTS assay,16 indicating an activity ranking of PG > 3 > 1 > 2.

INTRODUCTION Compartmentalization is a fundamental requirement in sustaining life.1 Lipid bilayer membranes of cells and intracellular organelles facilitate storage of biochemical energy to maintain ion homeostasis and electrochemical gradients.2 For example, the flow of H+ down a pH gradient across mitochondrial membranes drives F0 rotary ATP synthase to create biochemical energy (ATP);3 cellular ion concentration gradients are maintained by transmembrane protein channels,4 such as ATPase ion pump to mediate “active transport” processes, transporting ions against a concentration gradient fueled by ATP (Figure 1a). Development of supramolecular synthetic systems to facilitate transmembrane Cl− transport is often motivated by exploring new molecular carriers or channels for potential therapeutic applications and gaining insights on transport mechanism in lipid bilayer model membranes and cells.5 However, there has been less attention to the construction of transport systems capable of generating a transmembrane ion gradient across lipid bilayers,6 with no explicit example for anions. Unesterified free fatty acids have diverse biological functions, such as lipid metabolism, oxidative phosphorylation uncoupling, signal transduction, K+ channel activation, and as an energy source.7 Previously, Hamilton et al. have shown that addition of oleic acid (OA) to suspensions of phospholipid vesicles results in a decrease of intravesicular pH (pHin) and generates a transmembrane pH gradient.8 Herein, we report the use of synthetic anionophores to harness the H+ potential generated by fatty acid (FA) to drive H+/Cl− cotransport and establish a transmembrane Cl− gradient (Figure 1b). Prodigiosin (PG) is a natural product, known to be one of the most effective anionophores that can facilitate electro© 2019 American Chemical Society

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RESULTS AND DISCUSSION Methodology. As illustrated in Figure 2a, liposome-based assays using large unilamellar vesicles (LUVs) were prepared from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), loaded and suspended in the same concentration of NaCl (100 mM), buffered at pH 7.4 with HEPES (10 mM), encapsulated with either the pH-sensitive ratiometric fluorescent dye 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS, 1 mM) or the halide-sensitive fluorophore lucigenin (1 mM) to monitor the transport process. The fluorescence responses of HPTS and lucigenin were calibrated to pH and chloride concentration, respectively, for both inside the LUVs and after addition of detergent (see Supporting Information for details). Long-Chain Fatty Acids As Intravesicular Acidifiers. The mechanism of decreasing pHin on addition of FA is Received: March 4, 2019 Published: June 21, 2019 10654

DOI: 10.1021/jacs.9b02116 J. Am. Chem. Soc. 2019, 141, 10654−10660

Article

Journal of the American Chemical Society

than the volume of the bulk solution,18 this process results in a decrease of pHin only, thus establishing a transmembrane pH gradient. Based on this mechanism, approximately 25% of the total amount of FA will undergo H+ dissociation inside the LUVs (%H+in), assuming all FA molecules partitioned into the lipid bilayer membrane. The membrane partition coefficients (Kp) of long-chain FAs such as oleic and palmitic acids measured in EYPC LUVs (by the ADIFAB (acrylodan-labeled intestinal fatty acid binding protein)19 fluorescent probe) are extremely high, in the range of 105−106.20 This indicates that nearly all the FA molecules will partition into the lipid bilayer, with only 1−10 ppm remaining in the aqueous phase. The degree of ΔpHin was evaluated on addition of oleic acid, sodium oleate (NaOL), and palmitic acid (PA) to the LUVs monitored by the HPTS assay (Figure 3a), summarized in Table 1. Remarkably, the addition of OA at 2.5 μM gave 24% Hin+, almost exactly the figure of 25% predicted. While increasing the concentration of OA produced larger ΔpHin, the corresponding %H+in decreases to 19% and 14% at 5 and 10 μM, respectively. This can be attributed to the combined effects of spontaneous self-assembly of FA21 and the gradual decrease in pHin. The addition of OA, PA, and NaOL at the same concentration (5 μM) produced similar ΔpHin and %H+in. At pH 7.0, the ΔpHin appeared to be larger but the corresponding %H+in is lower due to the shift in deprotonation equilibrium of FA at lower pH. Fatty Acid Fueled Cl− Transport Monitored by Liposomal HPTS Assay. As shown in Figure 3b, the addition of anionophores 1−3 and PG readily dissipates the pH gradient generated by the prior addition of OA. In this liposomal assay, the dissipation of pH gradient must be accompanied by the efflux of Cl− to maintain charge balance across the lipid bilayer membrane. To demonstrate that the observed pH dissipation must proceed with Cl− efflux, CCCP (a protonophore) was added as a control, which resulted in no observable change in pHin, similar to adding DMSO as blank. Studies using different fatty acids and concentrations gave similar responses. These observations support the concept of

Figure 1. Illustrations of (a) an active transport process by an ATPase ion pump to generate a transmembrane ion gradient and (b) this work in using fatty acid as a chemical fuel to drive chloride transport and generate a transmembrane chloride gradient; (c) overview of compounds used in this study and the EC50 values of H+/Cl− cotransport activity of 1,16 2,14 3,11a and PG.12 EC50 of 2 was obtained in the presence of OA.

illustrated in Figure 2b.8 Long-chain FAs such as oleic acid have an elevated apparent pKa of ∼7.6 when partitioned into EYPC liposomes;17 therefore ∼50% of carboxylic acid headgroups remain as neutral (at pH 7.4) and can translocate (flip) to the interior leaflet, where approximately half of these FA molecules will deprotonate inside the LUVs. Given that the volume of solution encapsulated by the LUVs is much smaller

Figure 2. Schematic illustration of (a) fatty acid fueled transmembrane Cl− transport to generate a Cl− gradient of [Cl−]in < [Cl−]out in POPC LUVs (lipid concentration 0.1 mM, mean diameter 200 nm); (b) mechanistic insight of long-chain fatty acids as intravesicular acidifiers. 10655


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Journal of the American Chemical Society ΔEx =

RT [x ]out ln ΔF [x ]in

[H+]in [Cl−]in = [H+]out [Cl−]out

Table 1. Overview of ΔpHin, %H+in, and ΔEH+ upon Addition of Intravesicular Acidifiers (Figure 3a) and the Corresponding Predicted ΔECl−, pHin, and [Cl−]in at ΔECl− = ΔEH+ Calculated with Respect to the Observed ΔpHin after Addition of Fatty Acid

intravesicular acidifier/μM

ΔpHin

%H+ina

OA/2.5 OA/5 OA/10 PA/5 NaOL/5 OApH7d/5

−0.166 −0.261 −0.400 −0.247 −0.260 −0.302

24.3% 18.6% 13.6% 17.7% 18.5% 14.4%

predicted values at ΔECl− = ΔEH+

ΔEH+a/ ΔECl−b/ mV mV −10.3 −16.0 −24.3 −15.3 −16.3 −18.5

−0.27 −0.41 −0.60 −0.39 −0.42 −0.32

pHinc

[Cl−]inc/ mM

7.395 7.393 7.390 7.393 7.393 6.994

99.0 98.4 97.7 98.5 98.4 98.8

(2)

Curve fitting analysis was performed on the normalized HPTS ratiometric intensity to derive the transport kinetics (Table 2) based on an overall 100% transport toward net-zero potential equilibrium. The half-life (t1/2) and initial rate (kinitial, in %·s−1) values of all anionophores (with 5 μM OA added) gave similar trends to the previously reported H+/Cl− cotransport activity (PG > 3 > 1 > 2, Figure 1c). The t1/2 and kinitial values of 1 with different fatty acids added at 5 μM are nearly identical as expected. Using the calculated [Cl−]in at ΔECl− = ΔEH+, kinitial can be expressed as the change in intravesicular chloride concentration (Δ[Cl−]in, μM·s−1) as well as the Cl− transport in absolute terms (Cl− efflux per anionophore per second) to enable comparison between different concentrations of fatty acids and anionophores. Interestingly, the rate of Δ[Cl−]in remains similar for an anionophore with different concentrations of OA added, despite the different degree of pH gradient generated, indicating that the rate of H+/Cl− coefflux is largely unaffected by the transmembrane H+ potential (ΔEH+) between −10 and −24 mV. This is because the transport process is electroneutral (H+/Cl− cotransport); on the contrary, the transport rate of an electrogenic process is likely to be affected by different degrees of membrane potential, depending on the charge and directionality of the ion flux.23 Gratifyingly, the absolute rates of Cl− efflux per anionophore for 1 and 2 are comparable with different loading of an anionophore (0.01 versus 0.1 mol %); however there are significant differences for 3 and PG because the observed rates at the lower 0.01 mol% loading of anionophore are nearly at the maximum measurable rate for this assay (see Supporting Information Figures S30, S39, and S49). To further demonstrate this proof-of-concept prototype system, we performed the liposomal HPTS assay with the initial addition of 1 followed by multiple additions of OA as “fuel” to continuously drive Cl− transport against its concentration gradient (Figure 4). Multiple pulses of OA (at 300 s time intervals) were added at a lower concentration of 2.5 μM because it was demonstrated to be more efficient in establishing ΔpHin compared to adding OA at higher concentrations (Table 1). Anionophore 1 at 0.01 mol% loading was chosen for this study because the transport rate is not too fast, hence allowing the observation of pHin drop (reestablishing the transmembrane pH gradient) with every OA addition, and the transport process is nearly at ΔECl− = ΔEH+ after 300 s. The rate of pH dissipation decreases slightly over five cycles of OA addition along with pHin toward net-zero potential (as marked with green cross-symbols in Figure 4). The gradual decrease of pHin toward ΔECl− = ΔEH+ after each cycle of OA addition is due to the buildup of Cl− gradient (i.e., ΔECl−), hence larger ΔEH+. This is exemplified from the predicted values of pHin versus pHout and [Cl−]in versus [Cl−]out calculated over 10 cycles of OA additions (based on a theoretical %H+in of 25%) where the resulting pHout and [Cl−]out are largely unchanged (pH 7.40 and 100 mM) with pHin at 7.35 and [Cl−]in at 89.8 mM (see Supporting Information Table S1). Transmembrane Cl− Transport against Concentration Gradient Monitored by Liposomal Lucigenin

Figure 3. Plots of pHin monitored by HPTS fluorescence response: (a) added with different fatty acids (denoted in final concentration) at t = 60 s; errors shown as thin lines with shaded boundaries are SD from three independent measurements; (b) added with OA (5 μM final concentration) at t = 60 s, followed by different ionophores (denoted in mol% carrier:lipid molar percent) at t = 120 s.

upon addition of intravesicular acidifier

(1)

a Calculated H+ dissociation inside LUVs (%H+in) with respect to the total amount of acidifier and ΔEH+, based on ΔpHin. bPredicted ΔECl− at net-zero transmembrane potential. cCorresponding pHin and [Cl−]in at ΔECl− = ΔEH+. dMeasurements carried out at pH 7.0.

using FA as a chemical fuel to drive Cl− transport and generate a transmembrane chloride gradient as depicted in Figure 2a. The pH gradient generated by FA can also be expressed as the transmembrane electrochemical H+ potential (ΔEH+), using the Nernst equation (eq 1).22 The negative ΔEH+ (Table 1) is harnessed by an anionophore to drive the H+/Cl− coefflux toward net-zero transmembrane potential, where ΔECl− = ΔEH+ (with the assumption that H+ and Cl− have the same permeability in the presence of an anionophore), therefore generating a transmembrane chloride gradient of [Cl−]in < [Cl−]out (Figure 2a). Using the Nernst equation and the condition of ΔECl− = ΔEH+, we solved (i.e., predicted) ΔECl− along with the corresponding values of pHin and [Cl−]in at netzero potential equilibrium using eq 2, summarized in Tables 1 and 2. See Supporting Information for all calculation details and the “Solver” calculator constructed using MS Excel. 10656


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Table 2. Overview of Fatty Acid Fueled Transmembrane Chloride Transport of Prodigiosin (PG) and Anionophores 1−3 (0.01 or 0.1 mol% Carrier:Lipid Molar Percent) Monitored by HPTS Fluorescence Response (Figure 3b) initial rates of transmembrane Cl− efflux (kinitial) ionophore (conc./mol%)

fatty acid /μM

PG (0.01 | 0.1) 1 (0.01 | 0.1)

OA/5 OA/2.5 OA/5 OA/10 PA/5 NaOL/5 OApH7d/5 OA/2.5 OA/5 OA/2.5 OA/5

2 (0.01 | 0.1) 3 (0.01 | 0.1)

ΔpHin −0.252 −0.166 −0.260 −0.389 −0.248 −0.247 −0.300 −0.165 −0.260 −0.165 −0.255

| | | | | | | | | | |

a

−0.255 −0.164 −0.257 −0.388 −0.249 −0.250 −0.301 −0.160 −0.268 −0.163 −0.257

−

[Cl ]in /mM

t1/2b/s

%·s−1b

Δ[Cl−]in μM·s−1c

Cl−·ionophore−1·s−1c

| | | | | | | | | | |

5.94 | 3.40 64.6 | 11.1 83.9 | 14.6 130 | 23.7 84.3 | 14.0 87.2 | 15.4 65.0 | 7.98 83.8 | 14.9 113 | 21.6 11.4 | 3.92 15.2 | 4.34

11.9 | 20.6 1.16 | 8.15 0.732 | 5.70 0.456 | 3.50 0.764 | 6.03 0.760 | 5.17 1.55 | 10.0 0.803 | 5.91 0.529 | 3.93 7.16 | 18.4 5.19 | 18.3

−182 | −315 −12.3 | −85.2 −11.4 | −87.8 −10.3 | −78.6 −11.7 | −92.2 −11.4 | −78.1 −19.1 | −124 −8.38 | −61.7 −8.27 | −62.9 −74.9 | −195 −79.3 | −280

11 | 1.9 0.73 | 0.51 0.68 | 0.52 0.61 | 0.47 0.70 | 0.55 0.68 | 0.46 1.1 | 0.74 0.50 | 0.37 0.49 | 0.37 4.5 | 1.2 4.7 | 1.7

98.5 98.9 98.4 97.8 98.5 98.5 98.8 99.0 98.4 99.0 98.5

a

98.5 99.0 98.5 97.8 98.5 98.5 98.8 99.0 98.4 98.9 98.5

a Predicted [Cl−]in at net-zero transmembrane potential calculated with respect to the observed ΔpHin after addition of fatty acid. bHalf-life (t1/2) and initial rate (kinitial, in %·s−1) derived from curve fitting analysis on the normalized HPTS ratiometric intensity using the single- and doubleexponential functions, respectively. ckinitial, expressed as change in intravesicular chloride concentration (Δ[Cl−]in) and absolute Cl− efflux per anionophore per second. dMeasurements carried out at pH 7.0.

Figure 4. Plot of pHin monitored by HPTS fluorescence response, added with anionophore 1 at t = 60 s and OA (2.5 μM) at t = 120 s and at every 5 min time interval to a total of five additions. Errors shown as thin lines with shaded boundaries are SD from two independent measurements. Green cross-symbols mark the pHin values toward ΔECl− = ΔEH+ before OA addition. Solid red lines are fitted curves using a single-exponential function to calculate the initial rates of ΔpHin per second (as shown) after each cycle of OA addition.

Assay. Lucigenin was encapsulated within LUVs as a fluorescent probe to monitor [Cl−]in (Figure 5a). To validate this assay, NaNO3 (10 mM) was added in the presence of 1 to mediate Cl−/NO3− exchange,24 which gave the expected Δ[Cl−]in of ∼10 mM (i.e., [Cl−in] = 90 mM). When OA (10 μM) was added in the absence of an anionophore, an apparent decrease of [Cl−]in of ∼5 mM was observed. This artifact is due to the change in pHin interfering with the fluorescence response of lucigenin.25 Fortunately, larger Δ[Cl−]in was recorded in the presence of anionophores, with the expected transport activity of 1 < 3. Surprisingly, the observed [Cl−]in decreased to ca. 85 mM, a larger decrease than the predicted value of 97.8 mM based on the ΔpHin measured from the HPTS assay (Tables 1 and 2). The resulting Δ[Cl−]in of 15 mM corresponds to H+ dissociation inside the LUVs from 89% of total OA added. The chloride pumping action can also be reproduced with 10 cycles of OA addition in the HPTS assay (see Supporting Information Figure S70). Encouraged by this, we performed the experiment in a similar manner using the lucigenin assay with three, six, and 10 OA addition cycles. The highly potent

Figure 5. Plots of [Cl−]in monitored by lucigenin fluorescence response, added with anionophore 1 or 3 (0.01 mol%) at t = 60 s and (a) OA or NaNO3 (10 μM or 10 mM, respectively) at t = 120 s, average of two or three independent measurements fitted using a three-parameter asymptotic function; (b) OA (2.5 μM) at t = 120 s and at every 60 s time intervals, up to 10 additions; errors shown as thin lines with shaded boundaries are SD from two or three independent measurements; green cross-symbols are predicted [Cl−]in values at ΔECl− = ΔEH+ based on the theoretical %H+in of 25% (see Supporting Information Table S1).

anionophore 3 (0.01 mol%) was used for this study to allow OA addition at shorter time intervals to minimize photodecomposition of lucigenin.26 As shown in Figure 5b, a distinct step-down Δ[Cl−]in can be monitored over 10 cycles to achieve a Cl− gradient of [Cl−]in 65 mM | [Cl−]out 100 mM with corresponding calculated pHin 7.21 | pHout 7.40 at ΔECl− = ΔEH+ = 11 mV. The transmembrane Cl− gradient collapses upon addition of detergent to lyse the LUVs. As shown in 10657


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Journal of the American Chemical Society Figure 5a and b, the apparent [Cl−] upon addition of detergent to lyse the LUVs recovered to ∼95−100 mM; this is within error from the expected 100 mM [Cl−] (see Supporting Information for details). A liposomal leakage study using the self-quenching calcein27 fluorophore was also carried out to ascertain the stability of LUVs in this Cl− pumping system (see Supporting Information). Achieving a Δ[Cl−]in of ∼35 mM from 10 cycles of FA fuel addition (total of 25 μM OA) corresponds to an 83% efficiency ratio; this is comparable to the 89% attained from a single 10 μM OA addition as mentioned earlier. Furthermore, the corresponding calculated pHin 7.21 based on the generated Cl− gradient of 65 mM | 100 mM is lower than the observed pHin ∼7.32 from the HPTS assay (see Supporting Information Figure S70). Based on the above, and the significant difference between the experimental and predicted Δ[Cl−]in (at ΔECl− = ΔEH+, based on the reported mechanism of long-chain FAs as intravesicular acidifiers with a theoretical %H+in of 25%8) is evidence that supports the hypothesis that there may be other underlying mechanisms playing a role to facilitate Cl− efflux, in addition to the pH gradient generated by OA. Asymmetrical distribution of FAs between the inner/outer leaflets of LUVs in the presence of anionophores can potentially generate additional Cl− efflux, via an exchange mechanism between the carboxylate headgroups of FAs on the outer leaflet. Using the isotopically labeled [1-13C]-oleic acid, a liposome-based 13 C NMR study revealed that anionophore 3 can induce uneven distribution of OA headgroups in the lipid bilayer, which can be logically deduced to be biased toward the inner leaflet based on the membrane curvature, hence allowing another mechanism of Cl− efflux in addition to the H+/Cl− cotransport driven by the pH gradient. Asymmetry in the lipid bilayer will create microdomains and potentially alter the vesicles to become nonspherical (a hallmark of biological membranes).28 To gain insights on the shape/size properties of LUVs, dynamic light scattering (DLS) measurements were performed using the same liposomal conditions from the multiple OA addition transport studies. Interestingly, the narrow unimodal distribution profile of LUVs became broader after the addition of an anionophore and has more consistent size distribution/average when OA is added in the presence of an anionophore. This indicates that the LUVs became less spherical,29 which can be attributed to the asymmetry of FAs and the POPC distribution in the lipid layer. See Supporting Information Sections S8 and S9 for details and discussion. It is worth mentioning that 1400 times more NaNO3 or NaOH must be added (as external fuel) to generate the same Cl− gradient in this liposomal system, thus highlighting the superior efficiency of using FA as a chemical fuel to drive transmembrane transport. The advantage of using long-chain FAs as intravesicular acidifiers over adding external fuels arises from the basis of compartmentalization, in which the intravesicular volume is significantly smaller than the external bulk; therefore, less fuel is required when transmembrane H+ potential (i.e., chemical energy) is generated by affecting change in the intravesicular pH.

equilibrium of ΔECl− = ΔEH+, the H+/Cl− cotransport process was dissected to reveal that the actual Δ[Cl−]in is much higher than the predicted value (based on the ΔpHin generated by FA), due to the asymmetric distribution of the FA headgroups between the lipid bilayer leaflets of LUVs. Using this prototypical chloride pumping system, a transmembrane Cl− gradient of 65 mM | 100 mM was generated efficiently upon addition of 25 μM OA. It is expected that most reported synthetic anionophores can be used in this system; hence this work can potentially set in motion future developments in creating new ion pumping systems with applications in biosignaling and bioenergetics.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.9b02116. Calculator for solving net-zero transmembrane potential (XLSX) Details on methodology of liposome-based transmembrane transport studies, HPTS calibration, lucigenin calibration, background studies of fluorescent probes, and arithmetic calculations; all plots of fluorescence response including control studies and curve fitting analysis; details and results of calcein leakage; 13C NMR and DLS studies (PDF)

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AUTHOR INFORMATION

Corresponding Author

*[email protected] ORCID

Philip A. Gale: 0000-0001-9751-4910 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS P.A.G. thanks the University of Sydney and the ARC (DP180100612) for funding. We acknowledge Dr. Donald Thomas, Dr. Doug Lawes, and Dr. Adelle Amoore of the NMR Facility within the Mark Wainwright Analytical Centre at the University of New South Wales for NMR support.

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REFERENCES

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CONCLUSIONS In summary, we have shown in this minimalistic system the first example of using fatty acid as a chemical fuel to drive chloride transport and generate a transmembrane Cl− gradient. Using the liposomal HPTS and lucigenin assays along with interpretation of results based on the electrochemical potential 10658


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Fatty Acid Fueled Transmembrane Chloride Transport

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