Analysis of Ca2+-Binding Sites in the MthK RCK Domain by X-Ray Crystallography
Regulator of K+ conductance (RCK) domains form a conserved class of ligand-binding domains that control the activity of a variety of prokaryotic and eukaryotic K+ channels. Structural analysis of these domains by X-ray crystallography has provided insight
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Introduction Large-conductance Ca2+-activated K+ channels (BK channels) are found in a wide range of tissues, and play a critical role in linking K+ efflux to increases in cytoplasmic Ca2+ levels, thus tying Ca2+ signaling to electrical hyperpolarization of the cell membrane (1, 2). In electrically excitable nerve and muscle cells, this linkage provides an important feedback mechanism to allow for rapid repolarization of the membrane, promoting termination of action potentials and smooth muscle relaxation (3–7). While structural analysis of the mammalian BK channel can be technically complex and has so far yielded low-resolution structural information (8–10), the prokaryotic Ca2+-activated K+ channel, MthK, can be expressed and crystallized prodigiously, and has served as a model system for understanding gating mechanisms in these channels (11–16). MthK and BK channels are regulated by Ca2+ binding to a conserved cytoplasmic domain, known as the regulator of K+ conductance (RCK) domain (8, 9, 13, 14, 17–20). RCK domains are ubiquitous among prokaryotic and eukaryotic K+ channels and
Nikita Gamper (ed.), Ion Channels: Methods and Protocols, Methods in Molecular Biology, vol. 998, DOI 10.1007/978-1-62703-351-0_22, © Springer Science+Business Media, LLC 2013
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Frank J. Smith and Brad S. Rothberg
transporters, and control the activity of these proteins through the binding of cytoplasmic ligands such as nucleotides, Na+, Ca2+, Mg2+, and H+ (21–28). In MthK, channel activation occurs through Ca2+ binding to an octameric “gating ring” of RCK domains, which in turn is tethered to the pore-lining helices of the channel (13). Upon Ca2+ binding, the RCK domain undergoes a conformational change which is then translated to the pore facilitating K+ conduction, though the structural interactions that underlie this conformational change are not entirely clear (11, 14). Our approach toward understanding the mechanisms of the conformational change has involved screening to determine crystallization conditions that may yield new conformations of the RCK domain, with the goal of solving high-resolution structures that will reveal the details of chemical bonds that underlie stabilization of a range of conformations, in the presence and absence of Ca2+. Here, we describe a representative successful crystallization protocol, which enabled us to confirm the presence of newly determined Ca2+ binding sites in the RCK domain that underlie MthK channel activation.
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Materials All solutions are prepared using deionized, distilled water (ddH2O).
2.1 Plasmids and E. coli Strains
1. E. coli strain BL21(DE3), purchased from Stratagene, La Jolla, CA. 2. MthK RCK plasmid: MthK RCK domain cDNA (residues M107-A336 in MthK protein sequence) in pET21a vector with D184N mutation generated using QuickChange (Stratagene, La Jolla, CA). The MthK RCK domain sequence was followed by a thrombin recognition/cleavage sequence (amino acids LVPRGS) and C-terminal hexahistidine tag, and the coding region was codon-optimized for E. coli expression
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