Minako Hirano

Lipid bilayers at the gel interface for single ion channel recordings

Single-channel recording using artificial lipid bilayers is along with the patch-clamp technique a very powerful tool to physiologically and pharmacologically study ion channels. It is particularly advantageous in studying channels that are technically difficult to access with a patch pipet. However, the fragility of the bilayers and the difficulty to incorporate ion channels into them significantly compromises measurement efficiency. We have developed a novel method for forming artificial lipid bilayers on a hydrogel surface that significantly improves the measurement efficiency. Bilayers formed almost instantly (<1 s) and were able to incorporate various types of ion channel proteins within a short time (<30 s) enabling multichannel measurements. These results indicate that this method can potentially be applied to developing high-throughput screening devices for drug design.

Rearrangements in the KcsA Cytoplasmic Domain Underlie Its Gating

A change of cytosolic pH 7 to 4 opens the bacterial potassium channel KcsA. However, the overall gating mechanism leading to channel opening, especially the contribution of the cytoplasmic domain, remains unsolved. Here we report that deletion of the cytoplasmic domain resulted in changes in channel conductance and gating behavior at pH 4 without channel opening at pH 7. To probe for rearrangements in the cytoplasmic domain during channel opening, amino acid residues were substituted with cysteines and labeled with a fluorophore (tetramethylrhodamine maleimide) that exhibits increased fluorescence intensity upon transfer from a hydrophilic to hydrophobic environment. In all cases channel open probability (Po) was ∼1 at pH 4 and ∼0 at pH 7. Major increases in fluorescence intensity were observed for tetramethylrhodamine maleimide-labeled residues in the cytoplasmic domain as pH changed from 7 to 4, which suggests the fluorophores shifted from a hydrophilic to hydrophobic environment. Dipicrylamide, a lipid soluble quencher, reduced the fluorescence intensities of labeled residues in the cytosolic domain at pH 4. These results reveal that a decrease in pH introduces major conformational rearrangements associated with channel opening in the KcsA cytoplasmic domain.

A polysaccharide-based container transportation system powered by molecular motors

In living cells, the motor protein myosin, which is driven by ATP hydrolysis, intracellularly transports cargo such as vesicles and organelles 2] by moving along actin filaments. There have been many reports of how myosin can transport artificial cargoes such as polystyrene microspheres, 4] gold nanoparticles, and quantum dots. 7] Recently, cargo transportation systems powered by artificial nanomotors were actively studied. However, in these studies, the biomolecular or nanomotors are directly bound to the specific cargo. Therefore, these systems can be applied only to the delivery of certain types of cargo. Herein, we report the first container transportation system to be powered by biological motors. In this system, myosin is attached to a polysaccharide-based container that can hold a cargo. In fact, under physiological conditions, myosin binds to a container-like vesicle that holds a cargo. As the polysaccharide can form complexes with various cargoes such as carbon nanotubes and DNA, we envision that this novel container transportation system will expand the applicability of artificial intracellular transportation systems, including medically relevant procedures such as gene therapy. We have previously reported the very interesting “dynamic” properties of b-1,3-glucan polysaccharides, which are typified by schizophyllan (SPG). In nature, SPG adopts a triple-stranded helical structure (t-SPG), which dissociates into a single chain (s-SPG) upon dissolution in dimethyl sulfoxide (DMSO). The s-SPG chain can recover its original triple-stranded helix when DMSO is exchanged for water. These processes are referred to as denature (from t-SPG to sSPG) and renature (from s-SPG to t-SPG), respectively (Figure 1a). We found that b-1,3-glucans and their derivatives can act as 1D hosts that helically wrap nanomaterials such as carbon nanotubes, conjugated polymers, DNA, and gold nanoparticles, and allow these nanomaterials to be dissolved in water through the denature–renature process. Therefore, we selected SPG as the container for our system. A schematic representation of our novel container transportation system is shown in Figure 1b. The “cargo” is wrapped with the “container” and transported on the “rail” by “wheels”. We choose single-walled carbon nanotubes

Channels Formed by Amphotericin B Covalent Dimers Exhibit Rectification

Amphotericin B (AmB) is a widely used antifungal antibiotic with high specificity for fungi. We previously synthesized several covalently conjugated AmB dimers to clarify the AmB channel structure. Among these dimers, that with an aminoalkyl linker was found to exhibit potent hemolytic activity. We continue this work by investigating the channel activity of the dimer, finding that all channels comprised of AmB dimers show rectification. The direction of the dimer channel in the membrane depended on the electric potential at which the dimer channel was formed. On the other hand, only about half the monomer channels showed rectification. In addition, these channels were easily switched from a rectified to a nonrectified state following voltage stimulation, indicating instability. We propose a model to describe the AmB channel structure that explains why AmB dimer channels necessarily show rectification.

Single channel properties of lysenin measured in artificial lipid bilayers and their applications to biomolecule detection

Single channel currents of lysenin were measured using artificial lipid bilayers formed on a glass micropipette tip. The single channel conductance for KCl, NaCl, CaCl2, and Trimethylammonium-Cl were 474 ± 87, 537 ± 66, 210 ± 14, and 274 ± 10 pS, respectively, while the permeability ratio PNa/PCl was 5.8. By adding poly(deoxy adenine) or poly(L-lysine) to one side of the bilayer, channel currents were influenced when membrane voltages were applied to pass the charged molecules through the channel pores. Current inhibition process was concentration-dependent with applied DNA. As the current fluctuations of α-hemolysin channels is often cited as the detector in a molecular sensor, these results suggest that by monitoring channel current changes, the lysenin channel has possibilities to detect interactions between it and certain biomolecules by its current fluctuations.

Current Recordings of Ion Channel Proteins Immobilized on Resin Beads

Current ion channel current measurement techniques are cumbersome, as they require many steps and much time. This is especially true when reconstituting channels into liposomes and incorporating them into lipid bilayers. Here, we report a novel method that measures ion channel current more efficiently than current methods. We applied our method to KcsA and MthK channels by binding them to cobalt affinity gel beads with histidine tags and then forming a lipid bilayer membrane on the bead. This allowed channels to incorporate into the bilayer and channel currents to be measured quickly and easily. The efficiency was such that currents could be recorded with extremely low amounts of protein. In addition, the channel direction could be determined by the histidine tag. This method has the potential to be applied to various channel proteins and channel research in general.

Immobilizing Channel Molecules in Artificial Lipid Bilayers for Simultaneous Electrical and Optical Single Channel Recordings

There has been much interest in imaging single drug bindings to ion channel proteins while simultaneously recording single channel current. We developed an experimental apparatus for simultaneous optical and electrical measurement of single channel proteins by combining the single molecule imaging technique and the artificial bilayer technique. However, one major problem is that single molecule imaging of drug bindings is limited by the innate thermal diffusion of channel proteins in the artificial bilayer. Therefore, immobilizing channel proteins in the bilayers is imperative for stable measurements of channel-drug interactions. For future studies on channel-drug interactions, we describe here three different methods for simultaneous optical and electrical observation of single channels in which channel proteins are immobilized. (i) Membrane binding protein annexin V reduces the lateral diffusion of single channel proteins in a concentration-dependent manner. (ii) Channel proteins are immobilized by anchorage through a polyethylene glycol (PEG) molecule to the glass substrate. (iii) Channels immobilized on a gel bead can be directly incorporated into artificial bilayers.