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. Author manuscript; available in PMC: 2010 Feb 1.
Published in final edited form as: J Am Chem Soc. 2008 Dec 24;130(51):17274. doi: 10.1021/ja8082363

Membrane Transporters for Anions That Use a Relay Mechanism

Beth A McNally 1, Edward J O’Neil 1, Anh Nguyen 1, Bradley D Smith 1,*
PMCID: PMC2814302  NIHMSID: NIHMS86618  PMID: 19035637

Abstract

A new type of synthetic membrane transporter is described and shown to operate in vesicles by a relay mechanism. The transporter structure is a phosphatidylcholine derivative with a urea group appended to the end of its sn-2 acyl chain. The urea can bind a chloride ion at the membrane surface via hydrogen bonds and then relay it through the bilayer interior to an acceptor molecule located in the opposite membrane leaflet. Three phosphatidylcholine derivatives were studied and transport rates increased with transporter affinity for chloride. The results of various controls studies are consistent with an anion counter transport process using a relay mechanism and a kinetically active aggregate of two or four transporter molecules. Transport is inhibited if the transporter resides in only one leaflet of the membrane, if the bilayer is too thick, and if the counter anion is sulfate dianion. The expected favorable formulation properties of these amphiphilic compounds should facilitate efforts to transform them into tools for biomedical research and perhaps as therapeutic agents.

Synthetic bilayer membrane transporters are usually classified mechanistically as mobile carriers or as ion channels.1 A mobile carrier associates with a target ion to form a discrete supramolecular complex that diffuses across the membrane; whereas, an ion channel is a relatively immobile structure that spans the bilayer and allows a continuous flow of ions.2 In recent years there has been increased effort to design synthetic membrane transport systems for anions, especially Cl−.3 One of the long-term goals of this work is to create transporter replacement therapies that can alleviate the symptoms of diseases caused by diminished levels of endogenous Cl− transport (e.g., cystic fibrosis).4 The field of anion transport is still in its early stages with most published studies focusing on fundamental transport studies using model bilayer membranes. In terms of transporter designs, nearly all have been highly lipophilic compounds that partition strongly and non-selectively into any membrane.5 However, next-generation designs must begin to address the requirements for pharmaceutical success, including the following formulation features: (a) acceptable solubility in physiological solution, (b) appropriate cell targeting and subsequent membrane partitioning, (c) lengthy residence time in the apical plasma membrane of target cells. Suitably designed amphiphilic transporters are likely to exhibit these desirable properties, however, it is quite challenging to design amphiphilic transporters that operate by carrier or ion channel mechanisms. This quandary has prompted us to design a new type of membrane transporter that operates by a relay mechanism.6

A generalized picture of the relay transport process is shown in Scheme 1. The transporter structure is a phospholipid derivative with an ionophore appended to the end of its sn-2 acyl chain.7 The ionophore can bind an ion at the membrane surface and then relay it through the bilayer interior to an acceptor molecule located in the opposite leaflet.8 In this initial study, the ionophore is a simple urea group that can associate with Cl− via hydrogen bonds.9 The phosphatidylcholine derivatives 1–3 were synthesized in a few steps and high yield using established procedures.10 Transporter 1 contains a 4-nitrophenylurea group with a relatively high affinity for Cl−, transporter 2 contains a weaker binding 4-tert-butylphenylurea group, and carbamate derivative 3 is a control structure with very weak Cl- affinity.11

Scheme 1.

Scheme 1

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Relay mechanism for dimeric transporter aggregate.

The ability of compounds 1–3 to transport Cl− into vesicles was measured using a standard fluorescence quenching assay.12 Briefly, compounds 1–3 were pre-incorporated into separate samples of unilamellar vesicles composed of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC):cholesterol (7:3 molar ratio, diameter 200 nm) and encapsulating the chloride sensitive fluorescent probe lucigenin. Addition of NaCl to the vesicle dispersions induces Cl− influx and quenching of the lucigenin fluorescence. The traces in Figure 1 clearly show that transporter 1 is superior to 2, whereas control 3 is essentially inactive. This trend of stronger anionophore producing enhanced transport has been reported before with a separate class of mobile carriers for Cl− that also utilize urea groups.13

Figure 1.

Figure 1

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Fluorescence quenching due to Cl− influx. At 100 s, an aliquot of NaCl (25 mM final conc.) was added to vesicles encapsulating the chloride sensitive probe, lucigenin (1 mM) and NaNO3 (225 mM), T = 25 °C. The vesicle membranes were composed of POPC:cholesterol (7:3) and either 1, 2, or 3 (5 mol %).

In order to elucidate the transport mechanism, a series of additional experiments were conducted with the most effective transporter, 1. The first experiment demonstrated that replacing the intravesicle NaNO3 with an isomolar concentration of Na2SO4 produced a greatly diminished rate of Cl− influx (see Figure S1 in the SI). This is consistent with an anion exchange process; that is, significant Cl− influx can only occur if there is a corresponding counter anion efflux, which is greatly diminished with the heavily solvated SO42–.3

The relay mechanism in Scheme 1 implies that the transporter must reside in both leaflets of the bilayer. This condition was met in the initial experiment which employed vesicles with 1 pre-incorporated in the membrane. However, no Cl− transport was observed when the experiment was repeated with one variation, external addition of 1 to preformed vesicles (see Figure S2 in the SI for details). In this case, the polar lipid 1 inserts into the outer leaflet of the vesicle membrane (confirmed by UV absorption) and does not migrate to the inner leaflet. To be effective, transporter 1 needs to populate both sides of the bilayer membrane.

The dependence of observed Cl− influx rate constants (kobs) on transporter concentration was determined in two vesicle systems with membranes of different compositions and thickness (i.e., 1,2-dimyristoylphosphatidylcholine (DMPC):cholesterol (7:3) and the thicker POPC:cholesterol (7:3)). In both cases, the curves were non-linear (Figure 2) indicating that transport is mediated by kinetically active aggregates of 1. Furthermore, linear relationships are obtained for the two membrane compositions when kobs is plotted against [1]n, where n = 2 and 4, respectively.14 Thus, the transporter aggregate number is two for the DMPC:cholesterol membrane which is consistent with the slightly overlapped tail-to-tail dimer shown in Scheme 1. An aggregation of four in the thicker POPC:cholesterol membrane membrane suggests that a pair of transporters are in each leaflet as shown in Scheme 2. An increased transporter aggregation number in thicker membranes has been seen before with self-assembled pore systems.15

Figure 2.

Figure 2

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Rate constants (kobs) for Cl− influx at different concentrations of 1 in vesicles composed of DMPC:cholesterol (7:3) (ν) and POPC:cholesterol (7:3) (λ), T = 25 °C. Inset: linear relationships with [1]n, where n = 2 and 4, respectively.

Scheme 2.

Scheme 2

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Relay mechanism for transporter aggregate of four.

The final mechanistic study with 1 measured Cl− influx rates as a function of vesicle membrane thickness. Transport was monitored in vesicles composed of phospholipids with increasing acyl chain length, and thus increased membrane thickness.15,16 Figure S3 in the SI shows that increasing the acyl chain carbon number from 14 to 18 produced an incremental decrease in transport rate. Significantly, when the acyl carbon number was increased to 20 and above there was a dramatic drop to essentially zero transport. This membrane thickness threshold effect is consistent with the relay mechanism and not with the two alternatives.17 When the membrane is relatively thin, the transporters can effectively relay Cl− across the lipophilic core of the membrane as shown in Scheme 1 and Scheme 3. Once the membrane is thicker than the tail-to-tail aggregate in Scheme 2 (whose polar head groups are anchored to their respective membrane interfaces), there is a gap between the urea groups in each leaflet and the energetic barrier for Cl− relay becomes prohibitively high.

In summary, we report a new class of synthetic membrane transporters whose molecular structures are phospholipids with anionophores appended to the end of the sn-2 acyl chain. The current design uses urea groups to bind and transport Cl−, however, it should be possible to employ other molecular recognition units to produce transporters that are selective for other anions, as well as cations and neutral polar molecules. Mechanistic studies indicate that the transporters operate by a new and distinct membrane relay process. The expected favorable formulation properties of these amphiphilic compounds (e.g., as liposomes, micelles, etc) should facilitate efforts to transform them into tools for biomedical research and perhaps as therapeutic agents.

Supplementary Material

1_si_001

Acknowledgement

This work was supported by the NIH and the University of Notre Dame.

Footnotes

Supporting Information Available: Synthetic procedures, transport experiments and data. This material is available free of charge via the Internet at http://pubs.acs.org.

References

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Supplementary Materials

1_si_001
NIHMS86618-supplement-1_si_001.pdf (554.8KB, pdf)

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