Ion channels or pores

Key parameters obtained from single-channel currents are the conductance g, the lifetime T and the open probability Pg of the single ion channel or pore (Fig. 11.4b). The conductance g informs on the ability of channels and pores to transport ions and therefore often relates to the inner channel diameter (Section 11.3.3). The conductance g increases usually with increasing ionic strength toward saturation at gMAX (Section 11.3.6). The lifetime is the average time a single... 

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]. 

Determination of the pH profile of a synthetic ion channel or pore early on is important to focus the further, often time consuming in-depth characterization on the pH of relevant activity [19, 21]. pH sensitivity is usually introduced with acidic or basic functional groups with a pKa near the pH of interest. Synthetic ion channels and pores with multiple acids or bases in the ion-conducting pathway exhibit parabolic pH profiles (Fig. 11.6). With fractional activity normalized between minima and maxima (7 = 0 at pHmin, T = 1 at pHmax), characteristics such as pHmax,... 

Fig. n.8. Gating charges Zg determined for non-ohmic (i.e. voltagegated) synthetic ion channels or pores (c) in vaiinomycin polarized, doubly-labeled LUVs (a) with internal HPTS to measure changes in pH and external Safranin Oto measure depolarization (b) (compare Fig. 

The anion/cation selectivity describes the overall preference of synthetic ion channels or pores for either anion or cations [5, 8]. The anion/cation selectivity can be determined in BLMs using salt concentration gradients between cis and trans chamber (Fig. 11.9A and Fig. 11.4). With salt gradients, a current will flow without applied voltage. The direction of this zero current reveals the preferred movement of K+ or CU through the synthetic ion channel or pore. The voltage required to... 

The reversibility of binding to the bilayer can be measured in a so-called hopping experiment [3]. In this assay on intervesicular transfer, the activity of synthetic ion channels or pores is measured in LUVs as described (Section 11.2.2). Then, fresh LUVs loaded with fluorescent probes are added for a second time. Inactivity in this second round demonstrates irreversible, activity reversible binding of synthetic ion channels or pores to the bilayer membrane of the initially added LUVs. 

The method of choice to determine, under meaningful conditions, the location of synthetic ion channels and pores in bilayer membranes is fluorescence depth quenching (FDQ) [4, 11]. In this well developed but costly analysis, the position of a quencher in the lipid bilayer is varied systematically. Analysis of the dependence of the efficiency to quench a fluorescent synthetic ion channel or pore on the position of the quencher reveals transmembrane, central or interfacial location (Fig. 11.13b-d). 

Evaluation of the meaningfulness of results from less sensitive and less selective methods needs additional attention (e.g. attenuated total reflectance infrared (ATR-IR) or solid-state NMR spectrometry) [2]. Indirect insights from functional studies include support for transmembrane orientation (Fig. 11.13b) from parabolic dependence of the activity of synthetic ion channel or pore on bilayer thickness (Section 11.3.7) [56] and other readouts in support of operational hydrophobic matching. Flippase activity may provide some support for interfacial location (Section 11.3.7, Fig. 11.13d) [61, 62]. 

The dipeptide Ser-12-Pro-13 segment connecting the two helices exists as a bend or a hinge allowing the two helices to be oriented in an L-shape configuration. I o-13 is a pivot point of the bend between the two helices. In aqueous solution pardaxin exists as a tetramer. However, it spontaneously assembles in membranes to form voltage-gated ion channels or pores. 

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