Anal. Chem. 2009, 81, 3151–3154
Current Recordings of Ion Channel Proteins Immobilized on Resin Beads Minako Hirano, Yuko Takeuchi, Takaaki Aoki, Toshio Yanagida, and Toru Ide* Network Center for Molecular and System Life Sciences, Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka Suita, Osaka 565-0871, Japan 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. To measure ion channel current, a series of procedures must be performed including the construction of mutants, expression of channels in cells, extraction of these channels from the membrane, reconstituting them into proteoliposomes, and conducting electrophysiological analysis by the planar bilayer method1 or the patch clamp method. These procedures have been applied to KcsA and MthK channels, both of which are potassium channels whose crystal structures are known2,3 making them an ideal model for our study. Current techniques offer electrophysiological analysis of specific channels and controlled constitution of lipids and solutions. However, they also have disadvantages. For example, a high concentration of channel proteins are needed, the channel direction cannot be controlled, and it takes more than 1 day to reconstitute channels into liposomes using dialysis. In addition, efficiency of current measurement is low because the incorporation of channels into the artificial lipid membrane by the planar bilayer method is stochastic. This is quite a limitation when studying physiological channel proteins since channel incorporation must be very high. This is contrary to channel forming toxins * To whom correspondence should be addressed. Dr. Toru Ide, Network Center for Molecular and System Life Sciences, Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka Suita, Osaka 565-0871, Japan. E-mail:
[email protected]. Phone: +81-6-6879-4632. Fax: +81-6-68794634. (1) Ion Channel Reconstitution; Miller, C., Ed.; Plenum Press: New York, 1986. (2) Doyle, D. A.; Morais Cabral, J.; Pfuetzner, R. A.; Kuo, A.; Gulbis, J. M.; Cohen, S. L.; Chait, B. T.; MacKinnon, R. Science 1998, 280, 69–77. (3) Jiang, Y.; Lee, A.; Chen, J.; Cadene, M.; Chait, B. T.; MacKinnon, R. Nature 2002, 417, 515–522. 10.1021/ac900286z CCC: $40.75 2009 American Chemical Society Published on Web 03/18/2009
like R-hemolysin.4 Furthermore, when using the planar bilayer method, incorporation takes several tens of minutes. In this study, we describe a novel method that does not use reconstitution into a liposome. Our method offers high current measurement efficiency using low amounts of protein and can control the direction of the channels. EXPERIMENTAL PROCEDURES Constructs and Mutants. KcsA cloned into pQE-30 vectors including an N-terminal hexahistidine tag was a gift from Dr. Kubo. E71A, E71AL90C, and E71AG116C mutants were obtained using the QuickChangeTM site-directed mutagenesis kit (Stratagene). The E71A mutation is known to inhibit inactivation. MthK cloned into pQE-70 vectors including a C-terminal hexahistidine tag was a gift from Dr. Jiang. Protein Expression and Purification. pQE-30 plasmids containing the KcsA sequence were transformed into Escherichia coli XL1-Blue and overexpressed by the addition of isopropyl β-Dthiogalactopyranoside (IPTG) to a final concentration of 0.5 mM. Expressed channels were extracted from membrane fractions by 10 mM n-dodecyl β-D-maltoside (DM, Dojin). In the case of MthK sequence containing pQE-70 plasmids, the concentration of IPTG was 0.4 mM and DM was 50 mM. Binding Channel Proteins on Co2+-Based Metal Affinity Chromatography Gel Beads. Co2+ affinity gel beads (TALON Metal Affinity Resins, Clontech) were added to an extracted channel protein solution and incubated for 30 min at 4 °C in order to bind histidine tags to the gel beads. Nonspecific bound proteins were removed with wash buffer (5 mM DM, 20 mM Tris-HCl (pH 7.6), 100 mM KCl, 20 mM imidazol). Proteins were analyzed by SDS-PAGE for each purification step (Figure 1). Channel Incorporation and Electrophysiology. Figure 2A shows a schematic drawing of the experimental apparatus. A 35 mm culture dish was filled with recording solution (∼2 mL). Gel beads with channel proteins were set on the dish. Suspensions of 1 µL contained about 100 beads. To develop stable horizontal bilayers,5 a hole at the upper chamber was precoated with 50 mg/ mL asolectin in hexane and then coated with 50 mg/mL asolectin in hexadecane. The hole was about 100∼300 µm in diameter. After coated with asolectin, a thick lipid solution layer formed. The horizontal lipid layer was moved downward until gently contacting (4) Holden, M. A.; Bayley, H. J. Am. Chem. Soc. 2005, 127, 6502–6503. (5) Ide, T.; Yanagida, T. Biochem. Biophys. Res. Commun. 1999, 265, 595– 599.
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Figure 1. Binding steps of purified proteins on agarose beads. Lane 1, marker; lane 2, E. coli extract expressing KcsA; lane 3, the membrane fraction; lane 4, detergent-solubilized E. coli extract; lane 5, detergent-nonsolubilized E. coli extract; lane 6, nonbound proteins from Co2+ affinity beads; lane 7, nonspecifically bound proteins from beads; lane 8, confirmation of bound proteins (eluted with 300 mM imidazole).
a gel bead by using a micromanipulator. Contact between the bead and the membrane and the bilayer formation process could be easily seen with a microscope (see video in the Supporting Information). After a few minutes, a lipid bilayer membrane was spontaneously formed (Figure 2B). After several minutes, channel currents were measured. Channels could be incorporated even if a bilayer membrane that had already formed in solution contacted a bead (Figure 2C). However, this incorporation method needed careful operation. The bath solution was held at virtual ground, so voltage at the upper solution connected to a patch clamp amplifier by an Ag-AgCl electrode defined membrane potential. Recording solutions were 200 mM KCl with 10 mM Tris-MES buffer (pH 4.0) for KcsA and 200 mM KCl with 10 mM Tris-Hepes buffer (pH 9.0) for MthK. RESULTS AND DISCUSSION Figure 1 shows the results of purified KcsA with the histidine tag and its binding to Co2+ affinity gel beads via the histidine tag. Few channels were unbound (lane 6), indicating almost all extracted channel proteins bound to the gel beads. Nonspecifically bound proteins were removed with wash buffer (lane 7). To confirm that only KcsA bound specifically to the gel beads via the histidine tag, histidine was replaced by imidazol. As a result, only KcsA wild type was eluted (lane 8). KcsA mutants (E71A, E71AL90C, and E71AG116C) and MthK all bound specifically (results not shown). Bilayers formed on the beads as described in the Experimental Procedures. After the lipid layer contacted the bead, it became thinner, resulting in a rapidly formed lipid bilayer membrane (
