Koki Kamiya

Automated Parallel Recordings of Topologically Identified Single Ion Channels

Although ion channels are attractive targets for drug discovery, the systematic screening of ion channel-targeted drugs remains challenging. To facilitate automated single ion-channel recordings for the analysis of drug interactions with the intra- and extracellular domain, we have developed a parallel recording methodology using artificial cell membranes. The use of stable lipid bilayer formation in droplet chamber arrays facilitated automated, parallel, single-channel recording from reconstituted native and mutated ion channels. Using this system, several types of ion channels, including mutated forms, were characterised by determining the protein orientation. In addition, we provide evidence that both intra- and extracellular amyloid-beta fragments directly inhibit the channel open probability of the hBK channel. This automated methodology provides a high-throughput drug screening system for the targeting of ion channels and a data-intensive analysis technique for studying ion channel gating mechanisms.

Droplet Split-and-Contact Method for High-Throughput Transmembrane Electrical Recording

This paper describes the rapid and repetitive formation of planar lipid bilayers via a mechanical droplet contact method for high-throughput ion channel analysis. In this method, first, an aqueous droplet delivered in a lipid-in-oil solution is mechanically divided into two small droplets. Second, the two small droplets contact each other, resulting in the lipid bilayer formation. Third, an ion channel is immediately reconstituted into the bilayer and the transmembrane current signals are measured. By repeating this procedure, massive data sets of the channel signals can be obtained. This method allowed us to perform statistical analysis of α-hemolysin conductance (n = 256 within 30 min) and channel inhibition experiments by contacting different types of the droplets in a short time frame.

Droplet-based lipid bilayer system integrated with microfluidic channels for solution exchange

This paper proposes a solution exchange of a droplet-based lipid bilayer system, in which the inner solution of a droplet is replaced for the purpose of efficient ion channel analyses. In our previous report, we successfully recorded the channel conductance of alpha-hemolysin in a bilayer lipid membrane using a droplet contact method that can create a spontaneous lipid bilayer at the interface of contacting droplets; this method is widely used as highly efficient method for preparing planar lipid membranes. When only pipetting droplets of the solution, this method is highly efficient for preparing lipid membranes. However, the drawback of droplet-based systems is their inability to exchange the solution within the droplets. To study the effect of inhibitors and promoters of ion channels in drug discovery, it would be beneficial to conduct a solution exchange of droplets to introduce membrane proteins and to apply or wash-out the chemicals. In this study, we propose a droplet contact method that allows for the solution exchange of droplets via microfluidic channels. We experimentally and numerically investigated the bilayer stability with respect to exchanging flow rates, and then demonstrated a binding assay of an alpha-hemolysin using one of its blockers. The solution exchange in this system was conducted in less than 20 s without rupturing the membrane. We believe that the proposed system will enhance the efficiency of ion channel analyses.

Cell-sized asymmetric lipid vesicles facilitate the investigation of asymmetric membranes

Asymmetric lipid giant vesicles have been used to model the biochemical reactions in cell membranes. However, methods for producing asymmetric giant vesicles lead to the inclusion of an organic solvent layer that affects the mechanical and physical characteristics of the membrane. Here we describe the formation of asymmetric giant vesicles that include little organic solvent, and use them to investigate the dynamic responses of lipid molecules in the vesicle membrane. We formed the giant vesicles via the inhomogeneous break-up of a lipid microtube generated by applying a jet flow to an asymmetric planar lipid bilayer. The asymmetric giant vesicles showed a lipid flip-flop behaviour in the membrane, superficially similar to the lipid flip-flop activity observed in apoptotic cells. In vitro synthesis of membrane proteins into the asymmetric giant vesicles revealed that the lipid asymmetry in bilayer membranes improves the reconstitution ratio of membrane proteins. Our asymmetric giant vesicles will be useful in elucidating lipid-lipid and lipid-membrane protein interactions involved in the regulation of cellular functions.

A portable lipid bilayer system for environmental sensing with a transmembrane protein

This paper describes a portable measurement system for current signals of an ion channel that is composed of a planar lipid bilayer. A stable and reproducible lipid bilayer is formed in outdoor environments by using a droplet contact method with a micropipette. Using this system, we demonstrated that the single-channel recording of a transmembrane protein (alpha-hemolysin) was achieved in the field at a high-altitude (∼3623 m). This system would be broadly applicable for obtaining environmental measurements using membrane proteins as a highly sensitive sensor.

Towards combinatorial mixing devices without any pumps by open-capillary channels: fundamentals and applications

In chemistry, biology, medical sciences and pharmaceutical industries, many reactions have to be checked by transporting and mixing expensive liquids. For such purposes, microfluidics systems consisting of closed channels with external pumps have been useful. However, the usage has been limited because of high fabrication cost and need for a fixed setup. Here, we show that open-capillary channels, which can be fabricated outside a clean room on durable substrates and are washable and reusable, are considerably promising for micro-devices that function without pumps, as a result of detailed studies on the imbibition of open micro-channels. We find that the statics and dynamics of the imbibition follow simple scaling laws in a wide and practical range; although a precursor film obeying a universal dynamics appears in the vertical imbibition, it disappears in the horizontal mode to make the design of complex micro-channel geometry feasible. We fabricate micro open-channel devices without any pumps to express the green florescent protein (GFP) by transporting highly viscous solutions and to accomplish simultaneous chemical reactions for the Bromothymol blue (BTB) solution. We envision that open-capillary devices will become a simple and low-cost option to achieve microfluidic devices that are usable in small clinics and field studies.

A glass fiber sheet-based electroosmotic lateral flow immunoassay for point-of-care testing.

We have developed a quantitative immunoassay chip targeting point-of-care testing. To implement a lateral flow immunoassay, a glass fiber sheet was chosen as the material for the microfluidic channel in which the negative charge on the fiber surfaces efficiently generates the electroosmotic flow (EOF). The EOF, in turn, allows controllable bound/free separation of antigen/antibody interactions on the chip and enables precise determination of the antigen concentration. In addition, the defined size of the porous matrix was suitable for the filtration of undesired large particles. We confirmed the linear relationship between the concentration of analyte and the resulting fluorescence intensity from the immunoassay of two model analytes, C-reactive protein (CRP) and insulin, demonstrating that analyte concentration was quantitatively determined within the developed chip in 20 min. The limits of detection were 8.5 ng mL(-1) and 17 ng mL(-1) for CRP and insulin, respectively.

Preparation of Connexin43-Integrated Giant Liposomes by a Baculovirus Expression-Liposome Fusion Method

Connexin‐43 (Cx43) containing giant liposomes (GL) were prepared by a baculovirus expression–liposome fusion method. Recombinant budded viruses expressing Cx43 were prepared and then fused with GLs containing DOPG/DOPC at pH 4.5. Connexon formation on the GL membrane was observed by transmission electron microscope. Hydrophilic fluorescent dye transfers were observed through a Cx43‐mediated pathway not only between Sf9 (Spodoptera frugiperda) cells with Cx43 but also from giant Cx43 liposomes to Cx43‐expressing U2OS cells (human osteosarcoma cell). The functional connexin‐containing liposome is expected to be useful for cellular cytosolic delivery systems. The original orientation and function of Cx43 was maintained after integration into the liposomes. The liposome fusion method will create new opportunities as a tool for analysis of channel membrane proteins. Biotechnol. Bioeng. 2010;107: 836–843.

Parylene Mobile Microplates Integrated with an Enzymatic Release for Handling of Single Adherent Cells

An approach for manipulating single adherent cells is developed that is integrated with an enzymatic batch release. This strategy uses an array of releasable microfabricated mobile substrates, termed microplates, formed from a biocompatible polymer, parylene. A parylene microplate array of 10-70 μm in diameter can be formed on an alginate hydrogel sacrificial layer by using a standard photolithographic process. The parylene surfaces are modified with fibronectin to enhance cell attachment, growth, and stretching. To load single cells onto these microplates, cells are initially placed in suspension at an optimized seeding density and are allowed to settle, stretch, and grow on individual microplates. The sacrificial layer underneath the microplate array can be dissolved on a time-scale of several seconds without cytotoxicity. This system allows the inspection of selected single adherent cells. The ability to assess single cells while maintaining their adhesive properties will broaden the examination of a variety of attributes, such as cell shape and cytoskeletal properties.

Apoptotic marginal zone deletion of anti-Sm/ribonucleoprotein B cells

CD40L is excessively produced in both human and murine lupus and plays a role in lupus pathogenesis. To address how excess CD40L induces autoantibody production, we crossed CD40L-transgenic mice with the anti-DNA H-chain transgenic mouse lines 3H9 and 56R, well-characterized models for studying B-cell tolerance to nuclear antigens. Excess CD40L did not induce autoantibody production in 3H9 mice in which anergy maintains self-tolerance, nor did it perturb central tolerance, including deletion and receptor editing, of anti-DNA B cells in 56R mice. In contrast, CD40L/56R mice restored a large number of marginal zone (MZ) B cells reactive to Sm/ribonucleoprotein (RNP) and produced autoantibody, whereas these B cells were deleted by apoptosis in MZ of 56R mice. Thus, excess CD40L efficiently blocked tolerance of Sm/RNP-reactive MZ B cells, leading to production of anti-Sm/RNP antibody implicated in the pathogenesis of lupus. These results suggest that self-reactive B cells such as anti-Sm/RNP B cells, which somehow escape tolerance in the bone marrow and migrate to MZ, are tolerized by apoptotic deletion in MZ and that a break in this tolerance may play a role in the pathogenesis of lupus.