HPTS assay

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. 

The determination of pH profiles is no problem with the ANTS/DPX assay but more difficult with the pH-sensitive CF and HPTS assays [19, 21]. Determination in BLMs poses no problems and Na NMR spectroscopy should be possible as well. The determination of the ionic strength dependence in BLMs is routine and gives important information on the affinity of the ions to the channels (Section 11.3.6). However, the low conductance often observed at low ionic strength can make the experiments difiicult. For vesicle experiments with varied external ionic strength, osmotic stress can be avoided with high internal ionic strength and compensation of external under-pressure using sucrose [15, 18]. 

Supramolecular chemists without BLM-workstation can approximate the inner diameter of synthetic ion channels and pores by size exclusion experiments in LUVs. Differences in activity between the HPTS assay - compatible with all diameters - and the ANTS/DPX assay reserved for pores with diameter larger than 5 A and the CF assay for pores with larger than 10A can differentiate between ion channels and pores (Fig. 11.5). Larger fluorescent probes like CF-dextrans are available to identify giant pores or defects [26]. However, we caution... 

Supramolecular chemists without BLM-workstation can determine gating charges in polarized vesicles (Fig. 11.8), using the HPTS assay for synthetic ion channels and the ANTS/DPX assay for synthetic pores (Fig. 11.5) [7]. To polarize vesicles, an inside-negative Nernst potential is applied with a potassium gradient (Eq. 11.6), osmotically balanced with sodium, coupled with the potassium carrier valinomycin at intermediate concentrations sufficient for rapid potential buildup without immediate collapse, and monitored by an emission increase of the externally added probe safranin O (Fig. 11.8a and b). 

As discussed above, synthetic ion channels added to LUVs loaded with the pH-sensitive fluorophore HPTS and exposed to a pH gradient mediate the collapse of the latter by either H+ZK or OH /C1 antiport (Figs. 9B and 5). Sensitivity of the measured rate (e) to external cation (f) but not anion exchange (g) identifies cation selectivity (Fig. 11.9h) [30] sensitivity to external anion but not cation exchange identifies anion selectivity in LUVs [9, 10]. Anion/cation selectivity of synthetic ion channels determined in BLMs and LUVs are comparable ]9, 24]. Whereas the determination of anion/cation selectivities of synthetic pores in BLMs is as with synthetic ion channels and unproblematic, the HPTS assay is not applicable for this purpose. Indications on anion/cation selectivities of synthetic pores in LUVs can be obtained from comparison of CF and ANTS/DPX assays, because the CF assay reports activity for anion selective pores only ]8]. 

More difficult in BLMs, refined HPTS assays exist to address the special cases of selective transport of protons [11] and electrons [17] in LUVs. In the conventional HPTS assay (Fig. 11.5c), the apparent activity of proton channels decreases with increasing proton selectivity because the rate ofthe disfavored cation (M ) influx influences the detected velocity more than the favored proton efflux. Disfavored potassium influx can, however, be accelerated with the potassium carrier vaiinomycin (Fig. 11.8). Increasing activity in the presence of vaiinomycin identifies proton channels with H > K+ selectivity being at least as high as the maximal measurable increase (in unpolarized LUVs of course, compare Section 11.3.4). Important controls include evidence for low enough vaiinomycin concentrations to exclude activity without the proton channel (due to disfavored H+ efflux). The proton carrier FCCP is often used as complementary additive to confirm M+ > H+ selectivity (e.g. amphotericin B). 

As for selective electron transport in LUVs, many assays have been developed early on in studies directed toward artificial photosynthesis [37]. An elegant recent example applies the HPTS assay to active transport [17]. Namely, photoinduced electron transfer is detected as internal pH increase due to proton consumption during the reduction of a water-soluble quinone trapped together with HPTS within LUVs. 

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

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),... 

A modified version of the HPTS assay with external HPTS is used to measure endovesiculation (Figures 2c and 7). 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. 

Figure 13 Determination of ion selectivity (a) from cis-trans ion gradients in planar bilayer conductance experiments and (b, c) by external ion exchange in the HPTS assay Description of the resnlts in (d) anion and (e) cation selectivity sequences or topologies, the latter showing selectivities as a function of reciprocal ion radii or ion dehydration enwgies.

The identification of proton selectivity requires special attention because it can be difficult to detect and study in both planar bilayers and LUVs. In the HPTS assay, the apparent activity of proton transporters decreases with increasing H+ > M+ selectivity, because M+ antiport becomes more and more rate-limiting with increased selectivity. To solve this problem, vahnomycin can be added. " Recovered H+ transport activity in presence of the potassium carrier demonstrates H+ > K+ selectivity. Analogously, the proton carrier carbonyl cyanide 4-(trifiuoromethoxy)phenyIhydrazone (FCCP) has been used to confirm the M+ > H+ selectivity of, for example, amphotericin B. 

The leading techniques to monitor artificial photosynthesis in lipid bilayer membranes are the HPTS assay and the Hurst assay (Figure i9).9 65-67 jjj assays, an external electron donor with appropriate redox potential, usually a tertiary amine such as ETDA, is combined with an internal electron acceptor with appropriate redox potential. To convert photonic into chemical energy, reduction of the internal acceptor by the external donor must be uphill, that is, thermodynamically unfavorable. In the HPTS assay, the internal acceptor is a quinone, which consumes two protons during its reduction with two electrons to a hydro-quinone (Figure 19a). The resulting increase in internal pH in response to irradiation with light is detected by the pH-sensitive HPTS (Section 3). 

Figure 19 The HPTS assay and the Hurst assay to detect artificial photosynthesis, (a) In the HPTS assay, intenal quinone reduction with light produces a proton gradient, which is detected by the pH-sensitive HPTS. (b) In the Hurst assay, internal cobalt reduction with light is detected by a change in color, (c) Electrogenic and electroneutral photosynthesis can be discriminated in the Hurst assay by the sensitivity toward the proton carrier FCCP. (d) Hill analysis of dose response curves delivers the basic information on the photosystem.

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