Planar bilayer conductance

We have developed a series of models in which 8-12 monomers were packed in an antiparallel manner to form the channel. The kinetics of pore formation in liposomes (9) and the dependence of planar bilayer conductance on toxin... 

Abel, E. Maguire, G. E. M. Meadows, E. S. Murillo, O. Jin, T. Gokel, G. W., (1997) Planar bilayer conductance and fluorescent studies confirm the function and location of a synthetic sodium-ion-conducting channel in a phospholipid bilayer membrane J. Am. Chem. Soc. 119, 9061-9062. 

In this and the next section, standard techniques to study transport across bilayer and bulk membranes are briefly introduced. This section covers U-tube and planar bilayer conductance experiments, while Section 3 describes vesicle-based methods. 

Planar bilayer conductance experiments, particularly at the single-molecule level, are relatively time consuming and require substantial expertise in data analysis to properly... 

Figure 4 (a) Simplified expaimental setup and (b) typical result and data analysis of planar bilayer conductance experiments. 

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. 

More quantitative insights can be obtained in planar bilayer conductance experiments. With the Hille equation (2) ... 

Synthetic transport systems prefer to transport either anions or cations, and the preferred ions are transported according to a specific selectivity seqnence or topology (Figure 13). Both anion/cation selectivity and selectivity sequences/topologies can be determined with planar bilayer conductance experiments and with fluorogenic vesicles. 

In planar bilayer conductance experiments, salt concentration gradients between the cis and trans chamber are used to measure ion selectivity. With salt gradients, a current is flowing in the absence of an applied voltage. The potential needed to stop this current from flowing is... 

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.

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. 

In order to explore the possibility of whether one of the longer chain compounds could function as a pore, we prepared n-undecyl pyrogallol[4]arene. The capsule formed readily from six monomers having 11-carbon side chains and was isolated. It was then converted into the copper metal orgaific nanocapsule (MONC) by using the techniques developed in the Atwood laboratory [21]. The MONC was then added to an asolectin membrane and studied by the planar bilayer conductance method. Classic open-close behavior was observed for this compound at voltages in the range of +30 to +60 mV. The conductance data were recorded for potassium cation, which was present in a concentration of 450 mM [25]. 

Channel behavior was assessed for tetra-3-pentylpyrogallol[4]arene and tetra-4-heptylpyrogallol[4]arene by using the planar bilayer conductance technique. This method allows observation of open-close behavior in a bilayer membrane, which connotes channel activity. In the studies discussed here, soybean asolectin was used as the bilayer membrane. 

Tetra-4-heptylpyrogaUol[4]arene showed at least seven distinctive open states (different channels), some of which were of significant duration. Overall, this behavior was not considered to reflect a single, stable organization of the monomers within the bilayer, hi contrast, the planar bilayer conductance trace for tetra-3-pentylpyrogallol[4]arene showed a single open state that dominated the trace. 

---