A long-sought goal in structural biology has been the imaging of membrane proteins in their membrane environments. This goal has been achieved with electron crystallography1 in those special cases where a protein forms highly ordered arrays in lipid bilayers. It has also been achieved by NMR methods1 in proteins up to 50 kilodaltons (kDa) in size, although milligram quantities of protein and isotopic labelling are required. For structural analysis of large soluble proteins in microgram quantities, an increasingly powerful method that does not require crystallization is single-particle reconstruction from electron microscopy of cryogenically cooled samples (electron cryomicroscopy (cryo-EM))2. Here we report the first single-particle cryo-EM study of a membrane protein, the human large-conductance calcium- and voltage-activated potassium channel3 (BK), in a lipid environment. The new method is called random spherically constrained (RSC) single-particle reconstruction. BK channels, members of the six-transmembrane-segment (6TM) ion channel family, were reconstituted at low density into lipid vesicles (liposomes), and their function was verified by a potassium flux assay. Vesicles were also frozen in vitreous ice and imaged in an electron microscope. From images of 8,400 individual protein particles, a three-dimensional (3D) reconstruction of the BK channel and its membrane environment was obtained at a resolution of 1.7–2.0 nm. Not requiring the formation of crystals, the RSC approach promises to be useful in the structural study of many other membrane proteins as well.
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
The density maps are deposited in the Electron Microscopy Data Bank under accession numbers EMD-5114 and EMD-5121.
Raunser, S. & Walz, T. Electron crystallography as a technique to study the structure on membrane proteins in a lipidic environment. Annu. Rev. Biophys. 38, 89–105 (2009)
Frank, J. Three-Dimensional Electron Microscopy of Macromolecular Assemblies (Oxford Univ. Press, 2006)
Cui, J., Yang, H. & Lee, U. S. Molecular mechanisms of BK channel activation. Cell. Mol. Life Sci. 66, 852–875 (2009)
Neyton, J. & Miller, C. Discrete Ba2+ block as a probe of ion occupancy and pore structure in the high-conductance Ca2+-activated K+ channel. J. Gen. Physiol. 92, 569–586 (1988)
Horrigan, F. T. & Aldrich, R. W. Coupling between voltage sensor activation, Ca2+ binding and channel opening in large conductance (BK) potassium channels. J. Gen. Physiol. 120, 267–305 (2002)
Salkoff, L. et al. High-conductance potassium channels of the SLO family. Nature Rev. Neurosci. 7, 921–931 (2006)
Fodor, A. A. & Aldrich, R. W. Convergent evolution of alternative splices at domain boundaries of the BK channel. Annu. Rev. Physiol. 71, 19–36 (2009)
Jiang, Y. X. et al. Crystal structure and mechanism of a calcium-gated potassium channel. Nature 417, 515–522 (2002)
Chanda, B. & Mathew, M. K. Functional reconstitution of bacterially expressed human potassium channels in proteoliposomes: membrane potential measurements with JC-1 to assay ion channel activity. Biochim. Biophys. Acta 1416, 92–100 (1999)
Reers, M. J-aggregate formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential. Biochemistry 30, 4480–4486 (1991)
Galvez, A. et al. Purification and characterization of a unique, potent, peptidyl probe for the high conductance calcium-activated potassium channel from venom of the scorpion Buthus tamulus . J. Biol. Chem. 265, 11083–11090 (1990)
Miller, C., Latorre, R. & Reisin, I. Coupling of voltage-dependent gating and Ba++ block in the high-conductance, Ca++-activated K+ channel. J. Gen. Physiol. 90, 427–449 (1987)
Wang, L., Ounjai, P. & Sigworth, F. J. Streptavidin crystals as nanostructured supports and image-calibration references for cryo-EM data collection. J. Struct. Biol. 164, 190–198 (2008)
Wang, L., Bose, P. S. & Sigworth, F. J. Using cryo-EM to measure the dipole potential of a lipid membrane. Proc. Natl Acad. Sci. USA 103, 18528–18533 (2006)
Jiang, Q. X., Chester, D. W. & Sigworth, F. J. Spherical reconstruction: a method for structure determination of membrane proteins from cryo-EM images. J. Struct. Biol. 133, 119–131 (2001)
Grigorieff, N. FREALIGN: high-resolution refinement of single particle structures. J. Struct. Biol. 157, 117–125 (2007)
Long, S. B., Campbell, E. B. & MacKinnon, R. Voltage sensor of Kv1.2: structural basis of electromechanical coupling. Science 309, 903–908 (2005)
Long, S. B. et al. Atomic structure of a voltage-dependent K+ channel in a lipid membrane-like environment. Nature 450, 376–383 (2007)
Clayton, G. M. et al. Structure of the transmembrane regions of a bacterial cyclic nucleotide-regulated channel. Proc. Natl Acad. Sci. USA 105, 1511–1515 (2008)
Liu, G. et al. Position and role of the BK channel alpha subunit S0 helix inferred from disulfide crosslinking. J. Gen. Physiol. 131, 537–548 (2008)
Jiang, Y. X. et al. X-ray structure of a voltage-dependent K+ channel. Nature 423, 33–41 (2003)
Fodor, A. A. & Aldrich, R. W. Statistical limits to the identification of ion channel domains by sequence similarity. J. Gen. Physiol. 127, 755–766 (2006)
Yang, H. H. et al. Activation of Slo1 BK channels by Mg2+ coordinated between the voltage sensor and RCK1 domains. Nature Struct. Mol. Biol. 15, 1152–1159 (2008)
Ye, S. et al. Crystal structures of a ligand-free MthK gating ring: insights into the ligand gating mechanism of K+ channels. Cell 126, 1161–1173 (2006)
Zhang, Z. et al. A limited access compartment between the pore domain and cytosolic domain of the BK channel. J. Neurosci. 26, 11833–11843 (2006)
Bingham, J. P. et al. Synthesis of a biotin derivative of iberiotoxin: binding interactions with streptavidin and the BK Ca2+-activated K+ channel expressed in a human cell line. Bioconjug. Chem. 17, 689–699 (2006)
Ludtke, S. J., Baldwin, P. R. & Chiu, W. EMAN: semiautomated software for high-resolution single-particle reconstructions. J. Struct. Biol. 128, 82–97 (1999)
Stewart, A. & Grigorieff, N. Noise bias in the refinement of structures derived from single particles. Ultramicroscopy 102, 67–84 (2004)
Grigorieff, N., Beckmann, E. & Zemlin, F. Lipid location in deoxycholate-treated purple membrane at 2.6 angstrom. J. Mol. Biol. 254, 404–415 (1995)
Pettersen, E. F. et al. UCSF Chimera – a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004)
We thank N. Grigorieff and C. Xu for use of the Tecnai F30 microscope and help with data collection. We also thank S. Bian for sharing his BK purification methods, and Y. Yan for cell culture. Image processing and reconstruction made use of the Yale Biomedical High-Performance Computing Center.
Author Contributions L.W. and F.J.S. designed the experiments; L.W. purified and reconstituted the protein, made and imaged the cryo-EM specimens, and performed image processing and reconstruction. L.W. wrote custom software, with some algorithms contributed by F.J.S. L.W. and F.J.S. co-wrote the paper.
About this article
Cite this article
Wang, L., Sigworth, F. Structure of the BK potassium channel in a lipid membrane from electron cryomicroscopy. Nature 461, 292–295 (2009) doi:10.1038/nature08291
Preferred Formation of Heteromeric Channels between Coexpressed SK1 and IKCa Channel Subunits Provides a Unique Pharmacological Profile of Ca2+-Activated Potassium Channels
Molecular Pharmacology (2019)
Structure Determination by Single-Particle Cryo-Electron Microscopy: Only the Sky (and Intrinsic Disorder) is the Limit
International Journal of Molecular Sciences (2019)
Design and assembly of a chemically switchable and fluorescently traceable light-driven proton pump system for bionanotechnological applications
Scientific Reports (2019)
The Journal of General Physiology (2019)