Transport Experiments in Membranes

Book Chapter

Reference

Transport Experiments in Membranes

MATILE, Stefan, SAKAI, Naomi, HENNIG, Andréas

MATILE, Stefan, SAKAI, Naomi, HENNIG, Andréas. Transport Experiments in Membranes. In:

Steed, Jonathan W. & Gale, Philip A. Supramolecular Chemistry: From Molecules to Nanomaterials. Chichester : Wiley, 2012. p. 473-500

Available at:

http://archive-ouverte.unige.ch/unige:18369

Disclaimer: layout of this document may differ from the published version.

1 / 1

Transport Experiments in Membranes

Stefan Matile, Naomi Sakai and Andreas Hennig

Department of Organic Chemistry, University of Geneva, Geneva, Switzerland

Table of Contents

1 Introduction 03

2 Techniques 06

2.1 U-Tube 06

2.2 Planar Bilayer Conductance 08

3 Vesicle-Based Techniques 10

3.1 Fluorescence Spectroscopy with Labeled Vesicles 11

3.1.1 HPTS and Lucigenin Assay 14

3.1.2 CF and HPTS/DPX Assay 16

3.1.3 Assays with Membrane-Bound Probes 17

3.1.4 Assays with External Probes 18

3.2 Absorption Spectroscopy 19

3.3 Circular Dichroism Spectroscopy 20

3.4 NMR Spectroscopy 20

3.5 Miscellaneous 21

4 Functional Studies 21

4.1 Designing Experiments 22

4.2 Stoichiometry:

Hill Analysis and Undetectable Active Structures 24 4.3 Pore Diameter: Hille Analysis and Size Exclusion 27

4.4 Voltage Sensitivity 28

4.5 Ion Selectivity 30

4.6 pH Sensitivity 36

4.7 Ligand Gating and Blockage 37

4.8 Catalysis and Sensing 40

4.9 Photosynthesis 43

5 Epilogue 45

6 References 47

Keywords:

Ion channels, ion carriers, pores, sensors, photosynthesis, voltage gating, ligand gating, pH gating, vesicles, Hill analysis

Abstract:

In this contribution, we describe methods to characterize the activity of synthetic transport systems in translocating molecules across otherwise impermeable membranes. The first two sections introduce methods with bulk membranes (U-tube experiments) and planar or “black”

lipid membranes (BLMs), and close with a more comprehensive overview of methods involving liposomes, in particular large unilamellar vesicles (LUVs). The fourth section focuses on the application of these methods to the elucidation of the functional characteristics of synthetic transport systems. This includes sensitivity toward membrane composition, membrane potential, pH, anions, cations, molecular recognition (sensing), molecular transformation (catalysis) or light (photosynthesis).

1 INTRODUCTION

An essential aspect of living organisms lies in their ability to separate and protect the cellular interior from the surrounding environment, while retaining the ability to take in nutrients and excrete degradation products. Attracted by the challenge to understand, imitate or even partially replace nature’s machinery, chemists have not only begun to modify naturally occurring ion channels and pores but also to construct synthetic ion channels, carriers and pores from scratch, i.e. from abiotic scaffolds that are not known in nature.

Pertinent reviews detailing the structural variety of synthetic transport systems are available.^1-5 Recent developments in the field have shifted the focus from cation to anion transport,^4,5 and include novel supramolecular motifs to bind anions to the transport system such as the use of hydrogen bonds from catechols in transport system 1 or amidoxy donors in 2.^6,7 Another development is the utilization of anion- interactions in synthetic transport systems 3 and 4,^8,9 and anion-macrodipole interactions in oligourea macrocycles 5 (see Fig. 1 for structures).¹⁰ The particular challenge to overcome the Hofmeister bias in transporting very hydrophilic anions across the hydrophobic bilayer membrane (see Section 4.5) was recently addressed by hydrogencarbonate transport with prodigiosins 6.¹¹ Other developments include for example a novel “relay” mechanism, wherein an ion transported by 7 is passed on from a synthetic transporter on one side of the membrane to another transporter on the opposite site.¹² Particularly rewarding development may be the interaction of transition metals with organic ligands directing the self-assembly of synthetic ion channels and pores such as in 8 and 9,^13,14 the use of bioinspired motifs such as G-quartets in 10,¹⁵ and applications in artificial photosystems 4 or 9,^9,14 or to living cells.⁷

Herein, we mainly use the general term “synthetic transport systems” for systems which facilitate transport of ions / molecules across bilayer membranes by any mechanism except simple membrane disruption by detergents. Carriers are transport systems that take up their cargo on one side of the lipid membrane and diffuse to the opposite side where the transported cargo is released (Fig. 2b). Ion channels and pores are membrane-spanning structures which open up a continuous pathway to mediate transport without moving by themselves (Fig. 2a). The distinction between pore and ion channel is rather arbitrary and can refer to the size selectivity (Section 4.3) of the molecule or to its ability to transport organics or inorganics, respectively. It is important to note that differentiation between carrier and channel/pore mechanism can be

N

N N

O O

O O

CN NC

7 N O

O N

O O

N O O

N

O O

O O

N HN O O NH

HN O O NH

O

-O

O

-O

O^- O

-O O

N

N O NH

Bn NH N O O HN Bn

HN

O HN

O N N O

H NH H

N ON O O Si Si

O

HN O HN O

O HN O

N

N O

H N

H H

N ON O O Si Si O

O O

N O

OH HO

3

2 1

4

6

8 N N N N

O^- O

O^- O

N N N N N N O

O

N N N

N

N N O

O Zn

Zn Zn

9

10 O

NH O

O N H O

HN O N H O OH

OH N H O

N NH O

HO HO

H N O

NH NH

O

NO₂ HN O HN

O O PO

O O Me₃⁺N

-O

O O O

O O N

O

HO

OH Pd 5

Figure 1. Sample structures highlighting recent developments in the field of synthetic transport systems.

particularly challenging and that mixed modes may exist.^1,2,10 Furthermore, synthetic transport systems can act by different mechanisms depending on the experimental conditions, for example carriers and detergents can behave like ion channels and pores under certain conditions, and vice versa.^1,2,16 Other bioinspired modes of action include endovesiculation (Fig. 2c) and membrane fusion (Fig. 2d), which are both more complex processes and poorly explored from the point of view of supramolecular chemistry.^17-19

b)

a) c) d)

e) f) g) h)

Figure 2. Transport of ions and molecules (filled circles) across lipid bilayer membranes (orange) by a) ion channels and pores, b) carriers, c) endovesiculators and d) fusogens (empty blue ellipse), synthetic ion channels and pores are classified according to their a) unimolecular, e)

barrel-stave, f) barrel-hoop, g) barrel-rosette or h) micellar (toroidal) active structures.

Ion channel and pores can be unimolecular or supramolecular (Fig. 2).^1,2 Unimolecular ion channels and pores are hollow, most commonly helical oligomers that are sufficiently long to span a lipid bilayer membrane (2-4 nm). Since the synthesis of molecules of this size can be difficult and problems with correct folding are frequent, self-assembly of monomeric units driven by supramolecular interactions is often considered as the key solution. Vertical cutting of such a unimolecular “barrel” gives “barrel-stave” pores (e.g. 4) horizontal cutting “barrel-hoop” pores (e.g. 5), and both horizontal and vertical cutting gives “barrel-rosette” pores (e.g. 3, 8-10; Fig. 2).

Membrane pores that involve a significant perturbation of the membrane structure are commonly summarized as micellar or toroidal pores.

In this contribution, we will review methods to characterize the activity of synthetic transport systems in translocating molecules across otherwise impermeable membranes for beginners in the field. The following two Sections briefly introduce methods with bulk membranes (U-tube experiments) and planar or “black” lipid membranes (BLMs), and close with a more comprehensive overview of methods involving liposomes, in particular large unilamellar vesicles (LUVs). The fourth Section will then focus on how to apply these methods to elucidate the functional characteristics of synthetic transport systems. The text builds on an introduction to the topic in a previous book chapter published by Wiley,²⁰ and three essays on specific topics.^21-

23 The present version is overall simplified, covers different topics, and includes recent developments. Although a general introduction to the topic is aimed for, much material is naturally taken from personal experiences, and the authors wish to apologize in advance to all colleagues whose significant contributions to a vast field appears underappreciated.

2 TECHNIQUES

In this and the next Section, standard techniques to study transport across bilayer and bulk membranes are briefly introduced. This Section covers U-tube and planar bilayer conductance experiments, vesicle-based methods are described in Section 3.

2.1 U-Tube Experiments

U-tube experiments are attractive because of their simplicity and their clear-cut interpretation. They give a definite yes-no answer about the capability of synthetic molecules to transport hydrophilic molecules across a hydrophobic barrier.¹⁶ Thereby they unambiguously identify the ability of a molecule or supramolecule to function as an ion carrier. Ion carrier activity in the U-tube does, however, not exclude that the same molecule or supramolecule can also function as ion channel, pore or detergent in lipid bilayer membranes (and vice versa).

A conventional experimental set-up is shown in Figure 3. The originally U-shaped tubes are replaced today by small vials (usually ~2 mL total volume) with a separating plate in the middle and a connecting hole at the bottom. The bottom of a U-tube is filled with a hydrophobic

solvent of higher density than water (usually chloroform) containing the (mostly hydrophobic) synthetic transport system. This hydrophobic phase is referred to as bulk membrane, sometimes bulk liquid membrane. The bulk membrane is then covered at both sides with buffered aqueous phases at defined pH and ionic strength. In the simplest experiment, the “source phase” (or cis buffer) has a high concentration of the analyte and the “receiving phase” (or trans buffer) contains no analyte. For ion transport, this corresponds to a symport experiment, where high concentrations of salt MX in the cis buffer drives the co-transport of both cation M⁺ and anion X^- across the bulk membrane into the trans buffer. For cation antiport, M1X and M2X are added to cis and trans buffer, respectively. For anion antiport, MX₁ and MX₂ are used in the two aqueous phases.

source phase water

bulk membrane (chloroform) (trans) (cis)

receiving phase

X^-

+

picrate O₂N

O^- NO₂

NO₂

absorption / emission

time (s)

0 1 0.5 fractional activity Y

concentration EC₅₀ b)

N N

X^- +

lucigenin

concentration

c)

a) M⁺

M⁺ X^- ion carrier

Figure 3. Schematic set-up (a), typical result (b) and data analysis (c) of U-tube experiments.

Picrate salts are most convenient for the detection of cation/anion symport, which can be readily extended to the detection of cation antiport. The yellow picrate salts are added to the cis buffer, whereas a colorless salt is placed in trans buffer. During cation antiport, picrate is transported as hydrophobic counteranion of the carrier-cation complex across the bulk membrane, and the increase in absorption of picrate in trans buffer readily informs on the velocity of the process (Caution: Picrates are explosives, they should be handled in small portions only). Similarly classical countercations for the detection of anion antiport in U-tubes do not exist. However, most standard probes including lucigenin (see below) will work for this

purpose (unpublished results). Many alternative methods for the detection of U-tube activity are conceivable (ion selective electrodes or probes, etc). For the transport of charged molecules, standard probes such as 8-hydroxy-1,3,6-pyrenetrisulfonate (HPTS), 5(6)-carboxyfluorescein (CF) have been used directly to detect anion transport, p-xylene bis(pyridinium) bromide (DPX) or safranin O to detect cation transport (see below for structures).¹⁶

In all assays, aliquots are withdrawn from the receiving phase in defined time intervals and the concentration of the analyte is determined via a calibration curve (Figure 3b).

Alternatively, the concentration in the receiving phase can be continuously monitored when a pump and flow cuvette are available. For quantitative analysis, kinetic traces are measured for increasing carrier concentrations, and the obtained dose response curves are analyzed with the Hill equation to reveal the EC₅₀, that is the effective concentration needed to observe 50% of the maximal activity, and the Hill coefficient n (Figure 3c, Section 4.2).

The reproducibility of U-tube experiments is notoriously poor because the aqueous phases and the active interfaces where ion exchange processes occur cannot be stirred. As a result, experimental parameters such as temperature, stirring speed, and even size and position of the stirring bar need to be thoroughly controlled, and potential evaporation of solvent must be prevented. To improve reproducibility, mechanical mini-stirrers have been placed in the aqueous phases. Taking place within minutes, hours or days, transport in the U-tube is comparably slow.

2.2 Planar Bilayer Conductance

Planar bilayer conductance experiments are carried out by measuring the electrical potential and current between two chambers that are filled with buffer (Figure 4a).²⁴ The chambers can be arbitrarily named cis and trans chamber, whereas herein the chamber connected to the input electrode is cis and the chamber connected to the reference electrode is trans. The planar or “black” lipid membrane (BLM) is formed by painting a lipid solution over a tiny micrometer-sized hole connecting the two chambers. In absence of any ion transport systems, the lipid bilayer represents an insulator and no current can flow between the two electrodes.

Upon addition of a synthetic ion channel or pore to either cis or trans chamber, partitioning into the membrane occurs and current can flow between the two chambers. To facilitate partitioning, repulsive voltage can be applied or transport systems and lipids can be mixed before membrane formation. The magnitude of current is then recorded as a function of time and provides insights

into the electric properties of the functional supramolecule.

At high concentrations, “macroscopic” currents flowing through several channels and pores are observed. At low concentrations, single-molecule currents can be recorded and isolated functional supramolecules can be studied. The appearance of single-molecule currents is often considered as unambiguous experimental evidence for the existence of an ion channel or a pore, because only the very fast ion flux through ion channels and pores leads to sufficiently high currents to reach single-molecule sensitivity, while the diffusion of ion carriers is too slow to afford sufficiently high currents and detergents would simply disrupt the membrane.

reference electrode input

electrode

agar bridges

cis trans

sample

trans cis

current (pA)

time (ms) closed

single-channel current (I) / conductance (g)

t₁ t₂ t₃

t_1' t_3'

single-channel lifetime

open probability Po

X^- / M⁺ a)

b)

X^- / M⁺

ion channel / pore

open channel

t₄ t₅

t_2' t_4' t_5'

Figure 4. Simplified experimental set-up (a) and typical result and data analysis (b) of planar bilayer conductance experiments.

An idealized version of a simple single-channel current is shown in Figure 4b. Open and closed states of synthetic ion channels can be identified by high and low currents, and their typical on-off transitions appear in a stochastic manner. It is important to note that these transitions are invisible in ensemble measurements, i.e. in experiments with vesicles (Section 3) or at the “multichannel” level, and that they are by no means related to the macroscopic modes of gating discussed in Section 4.

Each individual open state is characterized by a single-channel current In and a respective lifetime tn. The distribution of individual lifetimes tn follows an exponential decay from which an “average” lifetime can be obtained indicating the kinetic stability of the functional supramolecule. Typical single-channel lifetimes are in the millisecond range but may vary from

the resolution limit at ~1 ms to more than minutes. The open probability Po considers the total times in which an ion channel or pore rests in an open or a closed state. Po thus reports on the thermodynamic stability of the active structure. The conductance g, which is obtained from the dependence of single channel currents on the applied voltage, informs on the transport efficiency of an ion channel or pore and directly relates to the inner diameter of the channel (see Section 4.3).

The single-channel trace drawn in Figure 4b is highly simplified. More often several different conductance levels are observed over time. These can either originate from several identical or different single channels in the membrane, including intermediate-states during the formation of the ion channel or pore. Subconductance levels appearing in an open channel originate from conformational changes or from binding of ligands or probes to the ion channel.

The latter has been used for sensing applications and structural studies of biological channels (Section 4.8).

Planar bilayer conductance experiments, particularly on the single-molecule level, are relatively time-consuming and require substantial expertise in data analysis to properly address the frequent occurrence of artifacts by meaningful controls and cautious data interpretation.

However, once mastered, planar bilayer conductance experiments provide the ultimate evidence for the existence of ion channels or pores and can offer deep mechanistic insights (Section 4).

The obtainable information is limited in the case of very labile (lifetimes below the resolution limit) and also in the case of very stable (“infinite” lifetime) ion channels and pores. It is important to realize that although the observation of single-channel currents is commonly considered as solid evidence for the existence of ion channels (although occasionally detergents like triton X-100 can produce “single-channel currents”),^1,2 failure to detect single-channel currents does not imply that the synthetic ion channel or pore does not exist. This may be traced back to incorrect lipid composition of the membrane, insufficient partitioning, inactivity at high ionic strength, insufficient transport rates, and so on.

3 VESICLE-BASED TECHNIQUES

An excellent compromise in simplicity and value of information is found in the use of large unilamellar vesicles (LUVs), which can be probed by various complementary methods such as fluorescence and NMR spectroscopy or ion-selective electrodes (LUVs have a diameter of

100-200 nm, SUVs 30-50 nm, GUVs > 300 nm, G stands for giant, S for small). The use of LUVs is recommended for supramolecular chemists entering the field, because they do not require specialized equipment, provide a rapid overview on various activities and offer numerous possibilities to dissect most of the relevant characteristics of ion channels, pores, and carriers.

Highly reproducible standard procedures to prepare LUVs of defined size and membrane composition exist and various tutorials can be found in the literature.²⁵ For routine use, LUVs prepared by freeze-thaw-extrusion techniques are usually preferable,¹⁶ whereas advanced dialytic detergent removal methods (rotating chamber) can be best when highest LUV quality is required.

Nature, purity and age of the phospholipids used influence LUV quality significantly.

3.1 Fluorescence Spectroscopy with Labeled Vesicles

Among the different methods to characterize supramolecular transmembrane transport systems, fluorescence methods are most straightforward to implement and feature a high sensitivity. This enables the rapid screening of numerous activities under varying conditions. To characterize the transmembrane activity of synthetic transport systems by fluorescence spectroscopy, vesicles need to be prepared which are labeled with one or more fluorescent probes. Labeled vesicles can be equipped with internalized (P_I) and membrane-bound (P_M) probes during their preparation or the fluorescent probe can be added externally to the pre- formed vesicles (Figs. 5 and 6). Examples of internal probes include 8-hydroxy-1,3,6- pyrenetrisulfonate (HPTS), 8-aminonaphthalene-1,3,6-trisulfonate (ANTS), 5(6)- carboxyfluorescein (CF), lucigenin or calcein (Fig. 7). These probes can be complementarily used to dissect certain specific transmembrane transport activities. For example the pH-sensitive dye HPTS can report on proton transport,^20,26 lucigenin is sensitive towards halide anions,²⁷ calcein toward calcium, and all probes can report on their own export.

To quantitatively evaluate any activity exerted by a synthetic molecule, the fluorescence of the probe is recorded in a time-dependent manner during addition of the molecule to be investigated. A typical series of experiments is shown in Fig. 5b. Commonly, a constant initial fluorescence I0 is recorded at the beginning of each experiment. Next, addition of various concentrations of the transport system leads to a change in fluorescence, which may be either an increase or a decrease depending on the type of experiment and the specific conditions. Lastly, a

a)

X₁^- X₂^- P = fluorescent probe

external internal

emission/absorption intensity

time (s)

I

I₀

I_MAX (or v_0(MAX))

I_MAX (or v_0(MIN)) fractional activity Y addition of

transporter

vesicles destroyed

(calibration) I_t

0 1 0.5 fractional activity Y

concentration transporter EC₅₀ (nM) P_I

P_M

P_E membrane bound

outside inside

M₁⁺ M₂⁺

ion transporter X^- + M⁺

symport

anion antiport

cation antiport

b)

c)

Figure 5. General configuration (a), typical result (b) and data analysis (c) of vesicle flux experiments.

detergent like triton X-100 is added at the end of each experiment to obtain the emission of the free fluorophore I. Traces of time-dependent fluorescence intensity I_t are normalized by subtraction of I₀ and division by (I I₀) such that the normalized fluorescence changes from 0 to 1 during the experiment. If necessary, the relevant range of intensities is then further narrowed down to IMAX and IMIN, the maximal and minimal values obtained without and with excess transporter before vesicle lysis. These values are set to 0 and 1 and define the fractional activity Y of the synthetic transport system. Alternatively, the activity may be quantified by determination of the initial rate v0. From a plot of Y against the concentration, the minimal and maximal detectable activities Y0 and Y as well as the EC50, and Hill coefficient n can be determined via fitting with the Hill equation (Fig. 5c, Section 4.2).

Fluorescence kinetics for transport in vesicles can show either continuous or all-or-none behavior. Continuous behavior refers to a gradual change in fluorescence intensity that reaches completion with time (Fig. 6a, dashed). All-or-none behavior is characterized by an initial burst that, however, stops halfway and never reaches completion (Fig. 6a, solid). All-or-none occurs when an overly hydrophobic transport system partitions into some vesicles only and can not transfer into the vesicles that remained untouched by the initial attack. Continuous behavior thus occurs with more hydrophilic transporters that are capable of reversible partitioning and

intervesicular transfer. Validity of this interpretation can be demonstrated by intervesiclular transfer experiments (Fig. 6b).²⁸ In this assay, new, fluorescently-labeled vesicles are added at the end of an experiment. More hydrophilic synthetic transport systems with continuous behavior will jump to the new membranes and cause an additional gradual increase in fluorescence (Fig. 6b, dotted). Too hydrophobic all-or-none transport systems will be incapable of intervesicular transfer (Fig. 6c).

application of gradient time (s) It

a)

time (s) It

d) time (s)

It

b) addition of

transporter

addition of transporter

addition of more vesicles

addition of transporter application of

gradient

e) ion transport is faster than detection limit

c) all-or-none behavior originates from irreversible

partitioning intervesicular

transfer

X

Figure 6. (a) Kinetics in vesicle transport experiments can show either continuous (dashed) or all-or-none (solid) behavior, originating (b) from the ability (dashed) or inability (solid, c) of the synthetic transport system to transfer intervesicularly into newly added or previously unoccupied

vesicles. (d) Inversion of sequence of addition from transport system to vesicles with ion gradients (dashed) to addition of ion gradients to vesicles with transport system (solid) shows

that kinetics commonly report on the formation of the active system rather than direct ion transport which is far beyond detection limits (e).

The gradual fluorescence change in transport experiments with labeled vesicles either directly reports on the kinetics of transmembrane ion transport or refers to the kinetics of formation of the functional system. To dissect these two possibilities, the sequence of addition can be reversed in transport assay where specific gradients can be applied. For example, the

sequence of addition in the HPTS assay (see Section 3.1.1) can be reversed from application of pH gradient before transporter addition to transporter addition before application of pH gradient.²⁹ If both curves are identical, ion transport is the rate-limiting step, but in most cases it has been observed that ion transport is much faster. If this is the case, addition of the transport system after application of the pH gradient will cause a relatively slow, continuous change in fluorescence (Fig. 6d, dotted) while application of the pH gradient after incubation with the transporter leads to a jump in fluorescence (Fig. 6e, solid). Such a burst of ultrafast ion transport far beyond the time resolution of standard fluorescence spectrometers is observed because the gradient will find a transport system ready for work, i.e. self-assembly of the transport system has already occurred without being seen by the fluorescent probe. The remaining part of the kinetic trace is ideally independent of the sequence of addition, since the usual complex cocktail of processes should have reached the same stage after the same time.

3.1.1 HPTS and Lucigenin Assay

The HPTS (or pyranine) assay is the ideal assay to characterize new synthetic transport systems because it is the least selective assay and produces a signal for most transport mechanisms (Fig. 7).²⁶ HPTS is a pH-sensitive fluorescent dye with a pK_a of around 7.3. The emission spectrum of HPTS is rather insensitive towards changes in pH owing to rapid and complete photodissociation in the excited state. The excitation spectrum exhibits two maxima at 404 nm for the protonated and at 454 nm for the unprotonated form with an isosbestic point at 416 nm. This permits the ratiometric (i.e., fluorophore concentration-independent) detection of pH changes in double-channel fluorescence measurements.

For the HPTS assay, LUVs are loaded with HPTS and exposed to a pH gradient. A transporter added to the system may catalyze the collapse of this gradient by transporting either proton or OH^-. The principle of the HPTS assay in detecting ion transport activity relies in the presumption that membrane transport is commonly electroneutral. This implies that translocation of positive or negative charges across the membrane is compensated by an additional transport of ions of the same charge but into opposite direction, e.g. H⁺/M⁺ antiport or OH/X antiport, or alternatively by transport of ions of opposite charge but into same direction, e.g. H⁺/X symport or OH/M⁺ symport (Fig. 7). Unidirectional symport is commonly thought to be less favorable because it leads to an osmotic imbalance. The HPTS assay will also report the efflux of HPTS

itself as pH gradient collapse, but this event can be readily identified by comparison of kinetics from assays dedicated to the detection of large pores and more dramatic damage (Section 3.1.2).

Failure to detect activity may be traced back to a very high selectivity of the synthetic transport system. For example, a highly selective K⁺ carrier would not transport a proton or hydroxide ion which would be necessary to detect its activity by the HPTS assay (see Section 4.4 for a modified version of the HPTS assay). Even less intuitively, the activity of a proton transporter may not fully be detected by the HPTS assay, because proton transport will stop owing to the opposing membrane potential that has built up (Section 4.5). The use of the HPTS assay to detect ion selectivity will be described later on (Section 4.5), as will be HPTS assays adapted to the detection of pores (Section 3.1.2), fusion (Section 3.1.3), endovesiculation (Section 3.1.4), photosynthesis (Section 4.9) and catalysis (Section 4.8).

N N

lucigenin O₃S

O₃S SO₃ OH

SO₃ O₃S

H₃N

SO₃

N

N HO O O

COO OOC

DPX ANTS CF

P_I

outside inside

ion transporter probe

efflux

anion antiport

cation antiport H⁺

OH^- P_I

X^-

M⁺

calcein HPTS

(pyranine)

O O

HO N OOC

OOC N COO

COO

COO

Figure 7. Selected examples for internal probes for functional and structural studies on ion transport in membrane with details for the HPTS assay.

The lucigenin assay reports specifically on the transport of halide anions.²⁷ Lucigenin is a fluorescent dye, which is moderately quenched by relatively high (10-100 mM) concentrations of halide anions (Cl, Br, I, but also SCN), presumably by an aborted electron transfer mechanism.³⁰ Asymmetric vesicles can be prepared, which contain halides either inside or outside of the vesicle. Addition of the anion transporter leads to equilibration of the halide concentrations by either X/Y antiport or M⁺/X symport. The transport can then be monitored

by either a decrease (initial high outside concentration of halide) or an increase (initial high inside concentration of halide) in the fluorescence of lucigenin.^12,27 The lucigenin assay has so far exclusively been used to determine Cl transport but may be similarly useful for Br and I or even SCN transport. Cross-reactivity patterns with exchanged internal and external ions may provide interesting information on the selectivity and transport mechanism of the membrane transporter. As with the HPTS assay, the lucigenin assay usually does not discriminate between anion transport and probe efflux. However, this difference can be made by comparison with dedicate probes for this purpose (see 3.1.2).

Whereas the lucigenin assay reports exclusively on anion transport, the calcein assay can report on cations, particularly calcium. Cation binding to the imidodiacetate ligands causes the required change in fluorescence. At higher concentrations, calcein also undergoes self- quenching and is used like CF to monitor probe efflux through large pores (see 3.1.2).

3.1.2 CF and HPTS/DPX Assay

The CF and the HPTS/DPX assay are probably the most popular assays to detect the activity of pores (Fig. 7). Both, CF and HPTS/DPX assay rely on high local concentrations inside the vesicle, such that the fluorescence of the probes is quenched. In the CF assay, self- quenching applies, while in the HPTS/DPX assay, HPTS is entrapped together with the quencher DPX.¹⁶ Addition of pores results in translocation of the fluorescent probe or quencher and thereby to a dilution. Consequently, quenching no longer applies and pore activity is signaled as fluorescence recovery. Both assays require a sufficiently large inner diameter of the pore such that HPTS, DPX or CF can pass through. HPTS/DPX assay detects both cation and anion selective pores, whereas CF is exclusively transported by anion transporters.

The HPTS/DPX assay is preferably used in place of the previous ANTS/DPX assay³¹ because the fluorescence intensity is much better. However, the ANTS/DPX assay remains preferable for the determination of pH profiles, because neither ANTS nor DPX is sensitive towards changes in pH in the physiologically relevant range around pH 7. Calcein is sometimes used in place of CF, both probes respond to parameters other than dilution (pH, cations). HPTS esters are routinely used as fluorogenic probes for esterase activity.^32,33 The transformative efflux of intravesicular HPTS acetate has been used to study synthetic catalytic pores (Section 4.8).³²

3.1.3 Assays with Membrane-Bound Probes

Membrane-bound probes, which are incorporated directly during vesicle preparation, are commonly fluorescently labeled phospholipids. These probes are mainly used to address more complex changes in membrane structure, thereby providing insights into the structure and mechanism of the transporters. Depending on the specific desired application, various fluorescently labeled phospholipids are available. Popular examples include boron dipyrromethane (BODIPY) probes as FRET acceptors,^34,35 DOXYL probes as quenchers for parallax analysis,³⁶ 7-nitro-benzofurazan (NBD) probes such as NBD-POPE for flip-flop assay,^10,37,38 and so on (Fig. 8).

P_M P_E

N N

H₂N NH₂ O

N O

DOXYL

N N B

F F

BODIPY

safranin O

NBD-POPE P O O

O O O

O O O

N ON

NO₂

NH

sodium dithionite non-fluorescent

micellar pore / flippase N

ON NH₂

NH sodium

dithionite

sodium dithionite

time (s) I_t 1

0.5 0.25

micellar pore / flippase

sodium dithionite sodium

dithionite

flip-flop no flip-flop

Figure 8. Selected examples for external and membrane-bound probes for functional and structural studies on ion transport in membrane with details for the NBD flip-flop assay.

NBD probes are often used to assay flip-flop.^10,37,38 Flip-flop refers to the reversible transversal diffusion of lipids from one leaflet to the other leaflet of a lipid bilayer membrane. In intact membranes, this transversal diffusion is very slow (t1/2 on the order of hours to days).

However, it can be accelerated by biological or synthetic flippases, a special class of membrane

transporters related to ion carriers. Alternatively, micellar pores are synthetic ion channels and pores with flippase activity and can thus be identified with flip-flop assay (Fig. 2; interfacial location of the transporter as second distinctive characteristic of micellar pores can be identified by fluorescence depth quenching experiments with DOXYL probes).

In the NBD assay, asymmetric vesicles are produced by addition of dithionite to the labeled vesicle solution, which reduces the nitro group of the outer layers NBD chromophore to an amine group (Fig. 8a). This external NBD reduction transforms the highly fluorescent push- pull fluorophore into a non-fluorescent chromophore. Because the lipid bilayer is impermeable towards dithionite, the NBDs at the inner surface are not affected. Reduction of the fluorescence intensity by 50% after dithionite addition supports the unilamellarity of vesicles and spherical bilayer membranes. Subsequent size exclusion chromatography gives vesicles where only the inner leaflet of the membrane carries fluorescent lipids. Incubation of the asymmetric vesicle with varying transporter concentrations for varying time intervals yields to partial equilibration of the inner and outer leaflet, the extent of which can be detected by addition of dithionite and fluorescence monitoring. Variations in the assay are required to address the kinetics of flip-flop, NBD reduction by dithionite, and the potential transport of dithionite by the added transport system.

BODIPY dyes are used in membrane fusion assays (Figs. 2d and 8). To characterize fusion, the mixing of the lipid and the mixing (and the leakage) of the content of vesicles has to be measured in the presence of the fusogen. For content mixing, the encounter of internal quenchers and fluorophores can be used (for example HPTS or ANTS and DPX, see 3.1.2). For lipid mixing, vesicles separately labeled with FRET-donors and FRET-acceptors can be used, and fusion is detected as increasing FRET for increasing fusogenic activity.¹⁸

3.1.4 Assays with External Probes

Assays with externally added probes include the potential-sensitive dye safranin O (Fig.

8). This cationic dye only loosely associates with an unpolarized vesicle membrane but binds more efficiently when an inside negative membrane potential is applied. This translocation into a more hydrophobic environment is accompanied with an increase in fluorescence and thereby reports on the extent of the applied membrane potential (see Section 4.4).³⁶

A modified version of the HPTS assay with external HPTS is used to measure

endovesiculation (Figs. 2c and 7).¹⁹ In this assay, HPTS is added to unlabeled LUVs. In the presence of an endovesiculator, external HPTS will be transported into the inner water pools of the produced multilamellar vesicles. This internalized HPTS will be insensitive to externally added quencher DPX and can be used as a measure for endovesiculation.

O O

O B

-O O

SO₃^- OH OH

HO

O B

-O

OH HO

O

SO₃^- OH

N NH

NH₂ O N N

HO OH

2-O₃PO O

5'-GMP G-quartet

K⁺ K⁺

N N

N H O N N R

H H

N NN H O

N N R H H N

N N H

O N N R H

H

N N N H

O N N R

H H

yellow red

Cs⁺

K⁺ Cs⁺

a)

b)

CD active CD silent

PV

CBA

Figure 9. Selected examples for (a) colorimetric and (b) CD probes that respond to covalent capture and ion-templated self-assembly, respectively.

3.2 Absorption Spectroscopy

Whereas the sensitivity of fluorescent probes is most attractive for biological applications, membrane-based sensing systems ultimately call for colorimetric probes, where high levels of important analytes such as glucose or cholesterol can be seen with the “naked eye”

as color change from yellow to red. The PV/CBA assay has been introduced recently for this purpose (Fig. 9a).³⁹ In this assay, LUVs are loaded with pyrocatechol violet (PV) to yield yellow vesicles. Then, 4-carboxyphenylboronic acid (CBA) is added extravesicularly. PV efflux through active pores is followed by spontaneous reaction of the catechol with CBA to afford the red colored boronate ester. Other colorimetric probes also exist that respond to changes on pH or transition metal coordination.

3.3 Circular Dichroism Spectroscopy

The development of probes that report the activity of ion channels and pores as changes in their CD (circular dichroism) spectra have been developed based on the ion selectivity of the templated assembly of GMP into G-quartets (Fig. 9b).⁴⁰ To detect the activity of pores with CD spectroscopy, vesicles are loaded with G-quartets. GMP efflux through active pores is reported as CD silencing due to G-quartet disassembly.

This simple technique is not applicable to ion channels that are too small or too selective to mediate the efflux of GMP. To detect the activity of ion channels with CD, the potassium selectivity of G-quartets can be used. For example, vesicles are loaded with GMP in the presence of potassium ions at concentrations above the dissociation constant (K_D) of G-quartets.

In the presence of cation transporters, external cation exchange from potassium to cesium results in CD silencing as a result of G-quartet disassembly within the vesicle in response to potassium ion efflux. Reversal of the direction of cation antiport with Cs-loaded vesicles and external potassium is even more attractive because the response to ion channel activity is chirogenic.

This method is one of the few methods where transport across and intactness of spherical membranes is simultaneously reported without additional effort (see Section 4.1).

G-quartet based CD probes have been adapted to the detection of osmotic pressure in vesicles. In this case, vesicle shrinking under hyper- and vesicle swelling under hypoosmotic pressure are detected as intravesicular G-quartet assembly and disassembly, respectively.

3.4 NMR Spectroscopy

NMR spectroscopy complements the use of fluorescence, absorption and CD spectroscopy in investigating supramolecular membrane transport systems. Naturally appealing to the supramolecular chemist, the usefulness of NMR assays should not be overestimated.

NMR is less straightforward to implement and sensitivity is limited. Nevertheless, it can be used to address questions about specific ion selectivities. In a conventional set-up, a paramagnetic compound that cannot cross the membrane is added externally to the vesicle solution. This leads either to line broadening or to a frequency shift of the external ions such that they can be distinguished from internal ions. If both signals can be seen, concerns about vesicle destruction or transport of the shift reagent become redundant.

In principal, transport of any ion, for which NMR active isotopes and a suitable shift reagent exist, can be investigated and in fact the use of NMR to observe transport of Na⁺, Cl, or Br has been suggested early on. Nonetheless, its routine use was mainly limited to ²³Na NMR spectroscopy, where external dysprosium triphosphate is used as a paramagnetic shift reagent to separate the chemical shifts of intra- and intervesicular Na⁺.⁴¹ Sodium flux is detected by line- width analysis or peak integration. More recently, Smith and Davis independently introduced the use of ³⁵Cl NMR with Co²⁺ as shift reagent,^42,43 and Davis and co-workers demonstrated that transport of hydrogencarbonate could be followed by using ¹³C NMR and Mn²⁺ as an external shift reagent.¹¹

3.5 Miscellaneous

Because of their advanced level of development, high sensitivity and broad applicability, fluorescence spectroscopy with labeled LUVs and planar bilayer conductance experiments are the two techniques of choice to study synthetic transport systems. The broad applicability of the former also includes ion carriers, but it is extremely difficult to dissect a carrier from a channel or pore mechanism by LUV experiments. However, breadth and depth accessible with fluorogenic vesicles in a reliable, user-friendly manner are unmatched by any other technique. Planar bilayer conductance experiments are restricted to ion channels and pores and are commonly accepted as substantial evidence for their existence. Extremely informative, these fragile single-molecule experiments can be very difficult to execute and interpret. Another example for alternative techniques to analyze synthetic transport systems in LUVs are ion-selective electrodes.

Conductance experiments in supported lipid bilayer membranes may be mentioned as well.

Although these methods are less frequently used, they may be added to the repertoire of the supramolecular chemist.

4 FUNCTIONAL STUDIES

The overall activity of a transporter is influenced by numerous parameters, which include buffer and membrane composition, membrane polarization, and osmotic stress to name only a few. The comparison of the intrinsic activity of different transporters on an absolute scale is nearly impossible for this reason. This is not further problematic because absolute activities are

probably the least interesting aspect of synthetic transport systems and arguably deserve little priority. What really matters is responsiveness to specific chemical or physical stimuli. This includes sensitivity toward membrane composition, membrane potential, pH, anions, cations, molecular recognition, molecular transformation (catalysis) or light. These stimuli-responsive, multifunctional or “smart” transport systems are attractive for use in biological, medicinal and materials sciences. Standard techniques to identify such unique characteristics rather than absolute activities or mechanistic details are outlined in this Section.

4.1 Designing Experiments

The study of transport processes in membranes is difficult because the relation between origins and phenotype are often complex. The design of meaningful experiments that can be interpreted with reasonable confidence is thus of highest importance. The key problem usually is to assess whether or not found characteristics are significant. To demonstrate significance, experiments designed to yield dichotomic behavior are ideal.¹⁰ Whereas isolate or parallel trends in complex systems can originate from less or completely unrelated processes, the inclusion of negative or positive control compounds that show opposite trends is of central importance for meaningful data interpretation. For instance, increases in activity, ion selectivity, Hill coefficient, gating charge, and so on, in response to changed conditions for all studied compounds can originate from changes in delivery to or partitioning into the membrane, not to speak of simply overlooked (but sometimes hard to identify) technical errors. The identification of controls that show dichotomic behavior under identical conditions can suffice to demonstrate that at least one of the two opposing trends is significant.

Before embarking into the search for significant characteristics, technical questions such as stirring, temperature control, intervesicular transfer (Section 3.1), delivery to the membrane, and so on, should be under control. Stirring of the vesicular suspension at vigorously controlled constant temperature during an entire transport experiment is absolutely necessary to avoid errors. The yield of delivery to the membrane determines the relevant concentration of the transporter in the membrane and thus often controls the apparent activity measured as EC₅₀ (Section 4.2). For delivery, a transporter is usually added as a 20 l drop of 100-times more concentrated “stock” solution in a polar organic solvent to 2 mL vesicle suspension. The transfer of the transporter from this drop of solvent to the vesicular membrane is one of the least

understood processes in transport experiments. From a technical point of view, it is highly important to find a solvent which provides efficient transfer to the membrane to ensure that a potentially efficient transporter is not simply overlooked. A potential approach to shed some light on this complex issue comprises an exhaustive solvent screening with the ultimate goal to elucidate which solvents help the transporter to cross the aqueous phase and reach the membrane.

Having established a rough guideline, solvent mixtures may be even more beneficial.

Additionally, non-destructive detergents have been recently applied to address delivery problems. For example, the highly viscous triton X-100 (and other members of the triton family) efficiently lyses membranes above the critical micelle concentration (cmc ~ 100 M) but is an excellent delivery agent when reaching the membrane in inactive, monomeric form.⁴⁴ Used at 8 mM as additive in a stock solution, injection of 20 l to 2 mL vesicular suspension will produce micelles that solubilize transporters for a short time but fall apart before reaching the membrane.

Other promising detergent additives for delivery include Span 80, a (beneficial) mixture of differently alkylated sorbitols, alkyl glucosides, cholate, etc.

Moreover, it is important to clarify basic questions before initiating the search for significant characteristics. For example, it is important to know if vesicles remain intact or are damaged during transport. This question can be either addressed with assays that demonstrate intactness (operational CD or NMR probes, see Sections 3.3 and 3.4), with meaningful comparisons of assays with different read-outs (better EC50’s in HPTS assay than in CF assay),⁸ or with dedicated experiments such as internal trapping.¹⁶ In the latter assay, transport activity is measured and compared among three different vesicle experiments. That are first, addition of all transporter in a single step to vesicles loaded with a fluorescent probe (Fig. 10c), second, stepwise addition of transporter in small portions well below the EC50 (Fig. 10b), and third, stepwise addition of transporter to vesicles loaded with a fluorescent probe plus a highly efficient inactivator (Fig. 10a).

Much higher activity in the absence of an internal inactivator (Fig 10b and 10c) than in the presence (Fig. 10a) suggests that the transporter has been irreversibly trapped in the vesicle without release of the probe. This indirectly demonstrates that transport occurs across the membrane of intact vesicles, because vesicle destruction would lead to inactivator dilution below the concentration at which the inactivator becomes inefficient. Whereas internal trapping assays demonstrate that vesicles remain intact during transport, they do not exclude that lipids are actively involved. This question can be addressed with flip-flop and related assays (Section

3.1.3).

time (s) I_t

PI

PI

P_I P_I

c) b)

a)

a) b) c)

Figure 10. Internal trapping assays can be used to prove that transport really occurs across the membrane of intact vesicles. Transporters are added in small portions well below EC50 to labeled vesicles with (a) or without (b) internal inactivators. Without inactivators, activity will gradually increase and reach the activity obtained when all transporters are added at once (b vs c). With inactivators, transporters are continuously trapped intravesicularly, and no activity is

observed even at high total concentrations (a vs c).

4.2 Stoichiometry: Hill Analysis and Undetectable Active Structures

Hill analysis is the most important technique to characterize synthetic transport systems.^45-48 For Hill analysis, the dependence of the fractional activity Y (Section 2.3.2) on the concentration cM of the monomer used to self-organize or self-assemble into active transport systems is measured (Fig. 11). The obtained dose response curve (or Hill plot, or cM profile) is analyzed by nonlinear regression using the Hill equation

Y = Y + (Y0 – Y) / (1 + (cM / EC50)ⁿ) (1)

where Y₀ is the minimal activity observed without transporter and Ythe maximal at saturation with excess transporter. The Hill analysis delivers the EC50 and the Hill coefficient n.

Already introduced before (Figs. 3 and 5), the EC50 is the effective monomer concentration needed to reach 50% activity. The smaller the better, the EC50 is a convenient empirical value to

compare the activity of different transporters. Many different processes can contribute to EC50’s, including delivery efficiency to the membrane (Section 4.1) and partitioning, self-assembly in solution, at the interface or in the membrane, reorientation in the membrane, intervesicular transfer, and so on. Dissection of the different contributions to EC50’s is possible, at least in part, by systematic modification of conditions, delivery additives, vesicles (membrane composition, etc), and compounds (hydrophobicity, etc). The determination of EC50’s as such is very easy in the U-tube and with fluorescent vesicles. In planar bilayer conductance experiments, EC50

measurements can be less straightforward, particularly in single-channel experiments.

Contributions from P_o are usually most important, also the conductance g matters much.

inactive precursor

active structure relative

energy 0

0.5

monomer concentration c_M

EC₅₀ n > 1

0 0.5

EC₅₀ n 1 a)

b)

cM

Y

stability active

structure short

lifetime long

denature stabilize Y

c)

n 1 n > 1

Figure 11. Hill plots are dose response curves that describe the dependence of activity on monomer concentration. Hill analysis can differentiate between unstable supramolecular active structures (a, n > 1; known stoichiometry, undetectable suprastructure) and stable supramolecular

or unimolecular active structures (b, n 1; unknown stoichiometry, detectable suprastructure).

Single channel-lifetimes () differentiate between labile and inert active structures, whereas open probabilities Po and Hill coefficients inform both on thermodynamic stabilities.

On first view, the Hill coefficient n reports on the cooperativity of the transport process.^45-48 Measured below EC50 to avoid artifacts from assay saturation, Hill plots with n > 1 show an upward curvature (Fig. 11a), Hill plots with n < 1 show a downward curvature (Fig.

11b), and Hill plots with n = 1 are linear. With n > 1, Hill coefficients correspond to the number

of monomers (or stable dimers, trimers, etc) in the active supramolecule, and EC50’s can represent its KD. With n 1, the situation is more complex.

To fully understand and correctly use all results from Hill analysis, it is crucial to always remember that the Hill equation only applies when the monomer concentration cM is much higher than the concentration of the aggregated suprastructure. In the case of n > 1, this means not only that the stoichiometry of the active supramolecule is known, it also means that the active supramolecular structure is unstable and thus a minority population. Namely, endergonic self- assembly implies that the active supramolecule is formed only by a small fraction of the total initial monomer concentration. Therefore, at concentrations relevant for function (around EC₅₀), the active supramolecule exists only in the presence of excess monomer, and classical, unselective methods such as IR or NMR become irrelevant because they will only report on the inactive monomers. Eventually claimed or requested structural support by NMR, X-ray, CD or other conventional techniques for n > 1 systems is thus intrinsically wrong, often seriously misleading. Applying higher concentrations would shift the equilibrium towards higher fraction of aggregates but would then introduce the problem of forming inactive supramolecular polymers. The challenging n > 1 situation with undetectable active structures resembles the familiar scene where only a few do the work while many stand around and watch (Fig. 11, a and c, energetically uphill formation of active structure). As it will be hard to see the workers in a zoom-free photo of this scene, n > 1 systems are undetectable by routine techniques. As they are the most common, efficient and useful ones, this is very important to understand and remember:

With n > 1, few work, active structures are thermodynamically unstable, supramolecules have known stoichiometry (n or a multiple of n) and are intrinsically undetectable in routine structural studies.

The complementary n 1 systems are either unimolecular or formed by stable supramolecules that erroneously appear as cM in the inapplicable Hill plots (Fig. 11, b and c). To stay within above picture, this situation reflects the perfect scene where everybody present really contributes to the work. The n 1 systems have unknown stoichiometry but are compatible with routine structural analysis. They are less desirable in practice because they tend to suffer from poor delivery and precipitation. Precipitation during delivery to the membrane is expressed in n

< 1 and incomplete Hill plots at high concentration, original kinetic traces might show bursts of high noise due to scattering. Biological model pores such as gramicidin A, melittin, hemolysin A, etc, act all as “invisible” n > 1 transporters.