ANTS/DPX assays

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]. 

The HPTS/DPX assay is preferably used in place of the previous ANTS/DPX assay because the fluorescence... 

Anion/cation selectivities and ion selectivity sequences from planar bilayer conductance and LUV experiments are comparable, at least qualitatively and to a certain extent. Anion/cation selectivities for large pores are usually weaker than for small channels. They can be estimated in LUVs by comparing results from appropriate assays with different selectivities such as the anion-selective CF assay and the nonselective ANTS/DPX assays. The HPTS or luci-genin assays are not applicable in this case because of unselective probe export. Ion selectivities depend significantly and in an understandable manner on conditions. For instance, pH-gated inversion of anion/cation selectivity with synthetic pores can be designed rationally. ... 

Many biological and synthetic transport systems have a hydrophobic external and charged internal surface. These ubiquitous internal acids or bases frequently account for pH sensitivity. For this reason, it is advisable to record pH profiles as early as possible during the characterization of the novel molecule and continue in-depth studies at opti-ntized pH. > The best technique to determine pH profiles is the ANTS/DPX assay, since both probe and quencher are not very pH-sensitive (Section 3.1.2). However, most assays are applicable as long as systematic corrections are applied. Determination of pH dependences in planar bilayer conductance experiments is straightforward. 

---