Abstract
Nanoscale electrical biosensors are promising tools for diagnostics and high-throughput screening systems. The electrical signal allows label-free assays with a high signal-to-noise ratio and fast real-time measurements. The challenge in developing such biosensors lies in functionally connecting a molecule detector to an electrical switch. Advances in this field have relied on synthetic ion-conducting pores1 and modified ion channels2,3,4 that are not yet suitable for biomolecule screening. Here we report the design and characterization of a novel bioelectric-sensing platform engineered by coupling an ion channel, which serves as the electrical probe, to G-protein-coupled receptors (GPCRs), a family of receptors that detect molecules outside the cell. These ion-channel-coupled receptors may potentially detect a wide range of ligands recognized by natural or altered GPCRs5,6, which are known to be major pharmaceutical targets7. This could form a unique platform for label-free drug screening8.
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References
Bayley, H. & Jayasinghe, L. Functional engineered channels and pores. Mol. Membr. Biol. 21, 209–220 (2004).
Bouzat, C. et al. Coupling of agonist binding to channel gating in an ACh-binding protein linked to an ion channel. Nature 430, 896–900 (2004).
Grutter, T. et al. A chimera encoding the fusion of an acetylcholine-binding protein to an ion channel is stabilized in a state close to the desensitized form of ligand-gated ion channels. CR Biol. 328, 223–234 (2005).
Ohndorf, U. M. & MacKinnon, R. Construction of a cyclic nucleotide-gated KcsA K+ channel. J. Mol. Biol. 350, 857–865 (2005).
Kristiansen, K. Molecular mechanisms of ligand binding, signaling, and regulation within the superfamily of G-protein-coupled receptors: molecular modeling and mutagenesis approaches to receptor structure and function. Pharmacol. Ther. 103, 21–80 (2004).
Armbruster, B. N., Li, X., Pausch, M. H., Herlitze, S. & Roth, B. L. Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand. Proc. Natl Acad. Sci. USA 104, 5163–5168 (2007).
Schlyer, S. & Horuk, R. I want a new drug: G-protein-coupled receptors in drug development. Drug Discov. Today 11, 481–493 (2006).
Thomsen, W., Frazer, J. & Unett, D. Functional assays for screening GPCR targets. Curr. Opin. Biotechnol. 16, 655–665 (2005).
Moreau, C. J., Prost, A. L., Derand, R. & Vivaudou, M. SUR, ABC proteins targeted by KATP channel openers. J. Mol. Cell. Cardiol. 38, 951–963 (2005).
Moreau, C. J., Jacquet, H., Prost, A. L., D'Hahan, N. & Vivaudou, M. The molecular basis of the specificity of action of KATP channel openers. EMBO J. 19, 6644–6651 (2000).
Dupuis, J. P., Revilloud, J., Moreau, C. J. & Vivaudou, M. Three C-terminal residues from the sulphonylurea receptor contribute to the functional coupling between the KATP channel subunits SUR2A and Kir6.2. J. Physiol. 586, 3075–3085 (2008).
Haider, S., Antcliff, J. F., Proks, P., Sansom, M. S. & Ashcroft, F. M. Focus on Kir6.2: a key component of the ATP-sensitive potassium channel. J. Mol. Cell. Cardiol. 38, 927–936 (2005).
Nichols, C. G. KATP channels as molecular sensors of cellular metabolism. Nature 440, 470–476 (2006).
Tucker, S. J., Gribble, F. M., Zhao, C., Trapp, S. & Ashcroft, F. M. Truncation of Kir6.2 produces ATP-sensitive K+ channels in the absence of the sulphonylurea receptor. Nature 387, 179–183 (1997).
Zerangue, N., Schwappach, B., Jan, Y. N. & Jan, L. Y. A new ER trafficking signal regulates the subunit stoichiometry of plasma membrane KATP channels. Neuron 22, 537–548 (1999).
Koster, J. C., Sha, Q., Shyng, S. L. & Nichols, C. G. ATP inhibition of KATP channels: control of nucleotide sensitivity by the N-terminal domain of the Kir6.2 subunit. J. Physiol. 515, 19–30 (1999).
Tucker, S. J. & Ashcroft, F. M. Mapping of the physical interaction between the intracellular domains of an inwardly rectifying potassium channel, Kir6.2. J. Biol. Chem. 274, 33393–33397 (1999).
Vivaudou, M. et al. Probing the G-protein regulation of GIRK1 and GIRK4, the two subunits of the K-ACh channel, using functional homomeric mutants. J. Biol. Chem. 272, 31553–31560 (1997).
McKinney, M., Miller, J. H., Gibson, V. A., Nickelson, L. & Aksoy, S. Interactions of agonists with M2 and M4 muscarinic receptor subtypes mediating cyclic AMP inhibition. Mol. Pharmacol. 40, 1014–1022 (1991).
Beguin, P., Nagashima, K., Nishimura, M., Gonoi, T. & Seino, S. PKA-mediated phosphorylation of the human KATP channel: separate roles of Kir6.2 and SUR1 subunit phosphorylation. EMBO J. 18, 4722–4732 (1999).
Light, P. E., Bladen, C., Winkfein, R. J., Walsh, M. P. & French, R. J. Molecular basis of protein kinase C-induced activation of ATP-sensitive potassium channels. Proc. Natl Acad. Sci. USA 97, 9058–9063 (2000).
Wess, J. G-protein-coupled receptors: molecular mechanisms involved in receptor activation and selectivity of G-protein recognition. FASEB J. 11, 346–354 (1997).
Li, J. H. et al. Distinct structural changes in a G protein-coupled receptor caused by different classes of agonist ligands. J. Biol. Chem. 282, 26284–26293 (2007).
Kuo, A. L., Domene, C., Johnson, L. N., Doyle, D. A. & Venien-Bryan, C. Two different conformational states of the KirBac3.1 potassium channel revealed by electron crystallography. Structure 13, 1463–1472 (2005).
Nishida, M., Cadene, M., Chait, B. T. & MacKinnon, R. Crystal structure of a Kir3.1–prokaryotic Kir channel chimera. EMBO J. 26, 4005–4015 (2007).
Long, S. B., Tao, X., Campbell, E. B. & MacKinnon, R. Atomic structure of a voltage-dependent K+ channel in a lipid membrane-like environment. Nature 450, 376–382 (2007).
Leifert, W. R., Aloia, A. L., Bucco, O., Glatz, R. V. & McMurchie, E. J. G-protein-coupled receptors in drug discovery: nanosizing using cell-free technologies and molecular biology approaches. J. Biomol. Screen. 10, 765–779 (2005).
Hosy, E., Derand, R., Revilloud, J. & Vivaudou, M. Remodelling of the SUR–Kir6.2 interface of the KATP channel upon ATP binding revealed by the conformational blocker rhodamine 123. J. Physiol. 582, 27–39 (2007).
Makarova, O., Kamberov, E. & Margolis, B. Generation of deletion and point mutations with one primer in a single cloning step. Biotechniques 29, 970–972 (2000).
Acknowledgements
We are grateful to E. Reuveny for PTX–S1, S. Seino for Kir6.2, D. Logothetis for M2, B. Kobilka and R. Stevens for prepublication β2 pdb files, and A. Dupuis, F. Fieschi and N. Hussy for useful discussions. A.-L. Prost and R. Derand with J.R. performed exploratory experiments on non-GPCR fusion proteins that helped guide our research strategy. S. Crouzy and S. Redon helped with molecular dynamics. This project, part of the European project Receptronics coordinated by M. Tartagni, and a postdoctoral fellowship to C.M. were funded by the Sixth Framework Program of the European Union (STREP NMP4-CT-2005-017114). Additional funds were provided by CEA (Commissariat à l'Energie Atomique; Programme Toxicologie Nucléaire Environnementale), CNRS and Université Joseph Fourier. J.D. was supported by a CEA doctoral studentship.
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C.J.M. and M.V. established the concepts of this study, analysed data and wrote the manuscript. C.J.M. performed the experiments with help from J.P.D. (TEVC) and J.R. (molecular biology, TEVC, outside-out recordings). K.A. helped with molecular modelling. M.V. supervised the project.
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Moreau, C., Dupuis, J., Revilloud, J. et al. Coupling ion channels to receptors for biomolecule sensing. Nature Nanotech 3, 620–625 (2008). https://doi.org/10.1038/nnano.2008.242
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DOI: https://doi.org/10.1038/nnano.2008.242