Fluorescence Spectroscopy with Labeled Vesicles

A conventional set-up to characterize synthetic ion channels and pores by fluorescence spectroscopy in vesicles uses large unilamellar vesicles (LUVs) that are labeled with one or more internal (Pi), external (Pe) or membrane-bound (Pm) fluorescent probes (Fig. 11.5a). Internal probes such as HPTS, ANTS or CF are most common to determine activity, external probes are used to reveal specific characteristics (e.g. the voltage-sensitive safranin O, Section 11.3.4), membrane-bound probes are mainly but not exclusively used for structural studies (Section 11.4). 

To determine activity, the change in fluorescence emission of internal probes in response to the addition of synthetic ion channels or pores to the LUVs is usually measured as a function of time at different concentrations (Fig. 11.5b). To calibrate the dose response, the emission lao of the free fluorophore is determined at the end of each experiment by, e.g. the addition of a detergent like triton X-100. The minimal and maximal detectable activities /min and Imax in the calibrated curves are then used to recalibrate for a fractional activity Y, and a plot of Y as a function of the concentration of the channel or pore yields the EC o, that is the effective channel concentration needed to observe Y = 0.5 (Section 11.3.2). ECso values depend on many parameters and can be further generalized (e.g. the dependence on LUV concentration reveals the PCsqjmin) at low, together with the maximal lipid/ pore ratio at high, LUV concentration, Section 11.3) [15]. With respect to BLMs, 

As in BLMs, failure to detect activity in LU Vs does not imply that the synthetic ion channel or pore does not exist. As a general rule, the difficulty of identifying activity in LUVs increases with increasing selectivity of either pore or assay (for the unrelated partitioning problem, see 4.1) [3, 7-11]. In other words and similar to the situation in BLMs, the most interesting synthetic ion channels or pores are the most difficult ones to detect. Tire HPTS assay is arguably the most useful assay to begin with because of its poor selectivity (Fig. 11.5c) [9-11]. 

HPTS is a pH-sensitive fluorophore (pk, 7.3) [6]. The opposite pH sensitivity of the two excitation maxima permits the ratiometric (i.e. unambiguous) detection of pH changes in double-channel fluorescence measurements. The activity of synthetic ion channels is determined in the HPTS assay by following the collapse of an applied pH gradient. In response to an external base pulse, a synthetic ion channel can accelerate intravesicular pH increase by facilitating either proton efflux or OH influx (Fig. 11.5c). These transmembrane charge translocations require compensation by either cation influx for proton efflux or anion efflux for OH influx, i.e. cation or anion antiport (Fig. 11.5a). Unidirectional ion parr movement is osmotically disfavored (i.e. OH /M or X /H symport). HPTS efflux is possible with pores only (compare Fig. 11.5b/c). Modified HPTS assays to detect endovesiculation (Fig. 11.1c) [16], artificial photosynthesis [17] and catalysis by pores [18] exist. 

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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 differentiate a carrier from a channel or pore mechanism by LUV experiments. However, the 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. Exflemely 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 is 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. 

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