Extramembrane control of ion channel peptide assemblies, using alamethicin as an example.

Ion channels allow the influx and efflux of specific ions through a plasma membrane. Many ion channels can sense, for example, the membrane potential (the voltage gaps between the inside and the outside of the membrane), specific ligands such as neurotransmitters, and mechanical tension within the membrane. They modulate cell function in response to these stimuli. Researchers have focused on developing peptide- and non-peptide-based model systems to elucidate ion-channel protein functions and to create artificial sensing systems. In this Account, we employed a typical peptide that forms ion channels,alamethicin, as a model to evaluate our methodologies for controlling the assembly states of channel-forming molecules in membranes. As alamethicin self-assembles in membranes, it prompts channel formation, but number of peptide molecules in these channels is not constant. Using planar-lipid bilayer methods, we monitored the association states of alamethicin in real time. Many ligand-gated, natural-ion channel proteins have large extramembrane domains. As these proteins interact with specific ligands, those conformational alterations in the extramembrane domains are transmitted to the transmembrane, pore-forming domains to open and close the channels. We hypothesized that if we conjugated suitable extramembrane segments to alamethicin, ligand binding to the extramembrane segments could alter the structure of the extramembrane domains and influence the association states or association numbers of alamethicin in the membranes. We could then assess those changes by using single-channel current recording. We found that we could modulate channel assembly and eventual ion flux with attached leucine-zipper extramembrane peptide segments. Using conformationally switchable leucine-zipper extramembrane segments that respond to Fe(3+), we fabricated an artificial Fe(3+)-sensitive ion channel; a decrease in the helical content of the extramembrane segment led to an increase in the channel current. When we added a calmodulin C-terminus segment, we formed a channel that was sensitive to Ca(2+). This result demonstrated that we could prepare artificial channels that were sensitive to specific ligands by adding appropriate extramembrane segments from natural protein motifs that respond to external stimuli. In conclusion, our research points to the possibility of creating tailored sensor or signal transduction systems through the conjugation of a conformationally switchable extramembrane peptide/protein segment to a suitable transmembrane peptide segment.

Dielectric spectroscopy for non-invasive monitoring of epithelial cell differentiation within three-dimensional scaffolds.

In this study, we introduce a cellular differentiation cellular model based on dielectric spectroscopy that characterizes epithelial differentiation processes. Non-invasive cellular monitoring was achieved within a three-dimensional microenvironment consisting of a cell-containing collagen I gel seeded onto microfabricated scaffolds. In this proof-of-concept investigation, Madin-Darby canine kidney cells were cultured within microfabricated, geometrically controlled scaffolds and allowed us to differentiate to hollow cyst-like structures. This transformation within the three-dimensional environment is monitored and characterized through dielectric spectroscopy while maintaining cell culture in vitro.

Transmission of extramembrane conformational change into current: construction of metal-gated ion channel.

Interaction with Fe(III) induces the reversible conformational switch of the extramembrane segment in the artificial receptor channel, which is transmitted into membranes as an increase in channel current (ion flux).

Dielectric relaxation in a water-oil-triton X-100 microemulsion near phase inversion.

A mixture of water (10 mM KCl), toluene and Triton X-100 (40:40:20 wt %) shows temperature-dependent phase inversion. The phase inversion has been studied by dielectric spectroscopy over a frequency range of 10 Hz to 1 GHz. At temperatures above about 37 degrees C, dielectric relaxation appeared around 10 MHz, which was due to interfacial polarization in a water-in-oil type emulsion. The dielectric relaxation drastically changed between 30 and 25 degrees C. With decreasing temperature, the intensity of dielectric relaxation increased steeply below 30 degrees C to attain a peak at 27 degrees C, where that change was associated with an increase in low-frequency conductivity by about three orders between 30 and 26 degrees C. The dielectric behavior has been interpreted in terms of interfacial polarization with a percolation model in which spherical water droplets, arranged in array in a continuous oil phase, are randomly connected with their nearest neighbors using water bonds.

Modifications of alamethicin ion channels by substitution of Glu-7 for Gln-7.

To evaluate the role of charged residues facing a pore lumen in stability of channel structure and ion permeation, we studied electrical properties of ion channels formed by synthesized native alamethicins (Rf50 (alm-Q7Q18) and Rf30 (alm-Q7E18)) and their analogs with Glu-7 (alm-E7Q18 and alm-E7E18). The single-channel currents were measured over a pH range of 3.5 to 8.7 using planar bilayers of diphytanoyl PC. The peptides all showed multi-level current fluctuations in this pH range. At pH 3.5 the channels formed by the four peptides were similar to each other irrespective of the side chain differences at positions 7 and 18. The ionization of Glu-7 (E7) and Glu-18 (E18) above neutral pH reduced the relative probabilities of low-conductance states (levels 1 and 2) and increased those of high-conductance states (levels 4-6). The channel conductance of the peptides with E7 and/or E18, which was distinct from that of alm-Q7Q18, showed a marked pH-dependence, especially for low-conductance states. The ionization of E7 further reduced the stability of channel structure, altered the current-voltage curve from a superlinear relation to a sublinear one, and enhanced cation selectivity. These results indicate that ionized E7 strongly influences the channel structure and the ion permeation, in contrast to ionized E18.