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Book Chapter Book Chapter The Characterization of Synthetic Ion Channels and Pores MATILE, Stefan, SAKAI, Naomi Abstract This chapter contains sections titled: Methods : Planar Bilayer Conductance, Fluorescence Spectroscopy with Labeled Vesicles. Characteristics : pH Gating, Concentration Dependence, Size Selectivity, Voltage Gating, Ion Selectivity, Blockage and Ligand Gating. Structural Studies : Binding to the Bilayer, Location in the Bilayer, Self-Assembly, Molecular Recognition. Reference MATILE, Stefan, SAKAI, Naomi. The Characterization of Synthetic Ion Channels and Pores. In: Schalley Christoph. Analytical Methods in Supramolecular Chemistry. Weinheim : Wiley, 2012. p. 711-742 DOI : 10.1002/9783527610273.ch11 Available at: http://archive-ouverte.unige.ch/unige:8374 Disclaimer: layout of this document may differ from the published version. 1 / 1 The Characterization of Synthetic Ion Channels and Pores Stefan Matile and Naomi Sakai Department of Organic Chemistry, University of Geneva, Geneva, Switzerland Table of Contents 1. Introduction 01 2. Methods 03 2.1. Planar Bilayer Conductance 04 2.2. Fluorescence Spectroscopy with Labeled Vesicles 07 2.3. Miscellaneous 09 3. Characteristics 10 3.1. pH Gating 10 3.2. Concentration Dependence 12 3.3. Size Selectivity 14 3.4. Voltage Gating 15 3.5. Ion Selectivity 17 3.6. Blockage and Ligand Gating 21 3.7. Miscellaneous 25 4. Structural Studies 26 4.1. Binding to the Bilayer 28 4.2. Location in the Bilayer 29 4.3. Self-Assembly 30 4.4. Molecular Recognition 31 5. Concluding Remarks 31 6. References 32 1. Introduction “Membrane transport is the quintessential supramolecular function.” The objective of this chapter is not to defend this statement by one of the pioneers of the field[1] but to provide a supramolecular chemist newly entering the field with an introductory guideline how to measure this function. Emphasis will be on transport by synthetic ion channels and pores,[2] summarizing lessons 1 learned during years of research on the topic. The term “synthetic” applies to ion channels and pores that are constructed from scratch with scaffolds that are not known to occur in nature; this covers neither the organic synthesis of biological scaffolds (e.g., -helices) nor chemically or biotechnologically modified biological channels and pores. We use the terms ion channels and pores can for the transport of inorganics and organics, respectively. Ion channels and pores can mediate this transport across lipid bilayers without moving by themselves and without disturbing the suprastructure of the membrane (Fig. 1b). Transporters that shuttle across the bilayer during transport are named ion carriers (Fig. 1a). Detergents differ from ion channels and pores because they disturb the suprastructure of the bilayer membrane. Differentiation between these modes of transport can be difficult because of analytical challenges as well as because they depend not only on suprastructures but also on experimental conditions (e.g., carriers and detergents can behave like ion channels and pores under appropriate conditions, and vice versa).[2] Synthetic ion carriers have been studied in detail for decades, whereas the design of supramolecules that operate by the more complex endovesiculation (Fig. 1c) or fusion (Fig. 1d) remain as future challenges. Mainly because of the enormous progress made in the synthesis of complex supramolecules, synthetic ion channels and pores can be considered as the topic of current interest in the field. a) b) c) d) channel / pore carrier Figure 1. Transport of ions and molecules (filled circles) across lipid bilayer membranes (grey) by a) carriers, b) ion channels and pores, c) endovesiculators and d) fusogens (empty black symbols). The structural classification of synthetic ion channels and pores differentiates between (macro)molecules and different classes of supramolecules.[2] Unimolecular synthetic ion channels 2 and pores are usually hollow, often helical (macro)molecules that are long enough to span common lipid bilayer membranes (2-4 nm). Virtual vertical cutting of this unimolecular “barrel” gives the “barrel-stave”, horizontal cutting the “barrel-hoop”, horizontal and vertical cutting the “barrel- rosette” motif (Fig. 2). barrel-hoop unimolecular barrel-stave barrel-rosette micellar ion channel/pore supramolecular ion channels/pores Figure 2. Structural classification of synthetic ion channels and pores. The barrel-stave architecture is a classic for both biological and synthetic ion channels and pores (Fig. 3). Whereas barrel-hoop motifs have received considerable attention, barrel-rosette ion channels and pores are just beginning to emerge. The more complex micellar (or “toroidal”) ion channels and pores are, on the one hand, different from detergents because the micellar defects introduced into the lipid bilayer are only transient. Micellar pores differ, on the other hand, from membrane-spanning (i.e., “transmembrane”) barrel-stave pores because a) they disturb the bilayer suprastructure and b) remain always at the membrane-water interface. A representative synthetic barrel-stave pore is shown in Fig. 3,[3,4] comprehensive collections of recently created structures can be found in pertinent reviews.[2] 2. Methods Two main methods to characterize synthetic ion channels and pores exist.[2] Conductance experiments in planar or “black” lipid membranes (BLMs) inform on the electric properties of the hollow interior of an ion channel or a pore.[5] Flourescence spectroscopy with labeled, usually large, unilamellar vesicles (LUVs) can inform on influx and efflux of ions and molecules through ion 3 channels and pores as well as on their suprastructures, depending on the fluorescent probes used.[3,6- 11] For a supramolecular chemist entering the field, LUVs may be preferable because they do not require specialized equipment and expertise, provide a rapid overview on activities and are sufficient for the identification of most of the relevant characteristics described in chapter 3. BLM conductance experiments require skilled hands and are time consuming. Moreover, the frequent occurrence of artifacts requires insightful controls and cautious data interpretation. However, BLM experiments are needed to prove the presence of an ion channel or a pore and can provide deeper mechanistic insights compared to LUVs. In the following, the basic principles of both methods are briefly introduced before the discussion of their application to characterize synthetic ion channels and pores in chapter 3. Figure 3. A representative synthetic barrel-stave pore. Molecular model with p-octiphenyl staves in grey, -sheet hoops in yellow, external fullerene ligands in gold and an internal -helix blocker in red (adapted from ref. 4). 2.1. Planar Bilayer Conductance A conventional set-up to characterize synthetic ion channels and pores in BLMs uses two chambers that are filled with buffer and named cis and trans (Fig. 4a).[5] Here, the cis chamber is arbitrarily connected to the input electrode and the trans chamber to a reference electrode. The BLM is formed in a tiny hole (micrometers) between cis and trans chamber. The BLM acts as an insulator. Without ion channels or pores, no current can flow between the two electrodes. In other words, phase transfer of anions X- and cations M+ in the buffer across the hydrophobic core of the lipid 4 bilayer is not possible. Synthetic ion channels and pores can be added to either cis or trans chamber. Repulsive voltage can then be applied to drive them into the BLM between the chambers. Although not desirable and more demanding to reproduce, poor partitioning can be bypassed by premixing of channels / pores and lipids before BLM assembly in the workstation (4.1). Anyway, once put in place, the current flowing through the synthetic ion channel or pore in the BLM is then recorded as a function of time. It provides insights on the electric properties of the interior of the functional supramolecule. single-channel a) b) exp lifetime 1 statistical analysis agar bridges counts input reference t electrode electrode n open channel t1 t2 t3 sample single-channel current (I) / closed conductance (g) current (pA) t1' t2' cis trans time (ms) statistical analysis open probability Po X- / M+ cis c)I1 = I2 = I3 = I4: d)(I1 = I3) (I2 = I4): e) (I1 = I4) (I2 = I3): 2 identical 2 different 1 single channel, single channels single channels 1 subconductance I2 I3 I2 I2 I3 I3 current (pA) current (pA) I (pA) current I I1 4 I1 I I1 4 trans 4 X- / M+ time (ms) time (ms) time (ms) Figure 4. Standard configuration of a BLM experiment (a) and analysis of single-channel recordings (b-e). In multichannel experiments, “macroscopic” currents flowing through an ensemble of synthetic ion channels and pores in the BLM are studied. The fast ion flux through ion channels and pores also allows for the resolution of currents flowing through single functional supramolecules. Because transport by ion carriers is too slow for this single-molecule resolution and detergents simply break the BLM, the appearance of single-molecule currents is often considered as the ultimate experimental 5 evidence for the existence of an ion channel or a pore. However, single-channel currents are not a prerequisite of ion channels.[5] Single-channel currents appear and disappear in a stochastic manner (Fig. 4b-e). It is important to realize that these typical on-off transitions between open and closed channels are conventional single-molecule phenomena that are, however, invisible in the average of the ensemble, that is,
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