Ion Channels pp 277-287 | Cite as

Analysis of Ca2+-Binding Sites in the MthK RCK Domain by X-Ray Crystallography

  • Frank J. Smith
  • Brad S. Rothberg
Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 998)

Abstract

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 toward mechanisms underlying ligand binding and channel gating, and thus the experimental strategies aimed at determining structures of liganded and unliganded forms of the domains may be useful in analysis of other ligand-binding domains. Here, we describe a basic strategy for crystallographic analysis of the RCK domain from the MthK channel, for determination of its Ca2+-bound structure.

Key words

Crystallization Cytoplasmic domain Calcium Binding site Potassium channel 
This is a preview of subscription content, log in to check access.

Notes

Acknowledgements

We wish to thank Karin Abarca-Heidemann, Andrew S. Thomson, Matthew M. Callaghan, and Elsie Samakai for technical expertise and helpful discussions. This work was supported by NIH grant R01 GM68523 to B.S.R.

References

  1. 1.
    Vergara C, Latorre R, Marrion NV, Adelman JP (1998) Calcium-activated potassium channels. Curr Opin Neurobiol 8:321–329PubMedCrossRefGoogle Scholar
  2. 2.
    Hille B (2001) Ion channels of excitable membranes, 3rd edn. Sinauer Associates, Inc., Sunderland, MAGoogle Scholar
  3. 3.
    Jaffe DB, Wang B, Brenner R (2011) Shaping of action potentials by type I and type II large-conductance Ca2+-activated K+ channels. Neuroscience 192:205–218PubMedCrossRefGoogle Scholar
  4. 4.
    Brayden JE, Nelson MT (1992) Regulation of arterial tone by activation of calcium-dependent potassium channels. Science 256:532–535PubMedCrossRefGoogle Scholar
  5. 5.
    Herrera GM, Heppner TJ, Nelson MT (2000) Regulation of urinary bladder smooth muscle contractions by ryanodine receptors and BK and SK channels. Am J Physiol Regul Integr Comp Physiol 279:R60–R68PubMedGoogle Scholar
  6. 6.
    Storm JF (1987) Action potential repolarization and a fast after-hyperpolarization in rat hippocampal pyramidal cells. J Physiol 385:733–759PubMedGoogle Scholar
  7. 7.
    Lancaster B, Nicoll RA (1987) Properties of two calcium-activated hyperpolarizations in rat hippocampal neurones. J Physiol 389:187–203PubMedGoogle Scholar
  8. 8.
    Yuan P, Leonetti MD, Pico AR, Hsiung Y, MacKinnon R (2010) Structure of the human BK channel Ca2+-activation apparatus at 3.0 A resolution. Science 329:182–186PubMedCrossRefGoogle Scholar
  9. 9.
    Yuan P, Leonetti MD, Hsiung Y, MacKinnon R (2011) Open structure of the Ca2+ gating ring in the high-conductance Ca2+-activated K+ channel. Nature 481:94–97PubMedCrossRefGoogle Scholar
  10. 10.
    Wu Y, Yang Y, Ye S, Jiang Y (2010) Structure of the gating ring from the human large-conductance Ca2+-gated K+ channel. Nature 466:393–397PubMedCrossRefGoogle Scholar
  11. 11.
    Ye S, Li Y, Chen L, Jiang Y (2006) Crystal structures of a ligand-free MthK gating ring: insights into the ligand gating mechanism of K+ channels. Cell 126:1161–1173PubMedCrossRefGoogle Scholar
  12. 12.
    Dong J, Shi N, Berke I, Chen L, Jiang Y (2005) Structures of the MthK RCK domain and the effect of Ca2+ on gating ring stability. J Biol Chem 280:41716–41724PubMedCrossRefGoogle Scholar
  13. 13.
    Jiang Y, Lee A, Chen J, Cadene M, Chait BT, MacKinnon R (2002) Crystal structure and mechanism of a calcium-gated potassium channel. Nature 417:515–522PubMedCrossRefGoogle Scholar
  14. 14.
    Pau VP, Smith FJ, Taylor AB, Parfenova LV, Samakai E, Callaghan MM, Abarca-Heidemann K, Hart PJ, Rothberg BS (2011) Structure and function of multiple Ca2+-binding sites in a K+ channel regulator of K+ conductance (RCK) domain. Proc Natl Acad Sci U S A 108:17684–17689PubMedCrossRefGoogle Scholar
  15. 15.
    Niu X, Qian X, Magleby KL (2004) Linker-gating ring complex as passive spring and Ca(2+)-dependent machine for a voltage- and Ca2+-activated potassium channel. Neuron 42:745–756PubMedCrossRefGoogle Scholar
  16. 16.
    Hou S, Xu R, Heinemann SH, Hoshi T (2008) Reciprocal regulation of the Ca2+ and H  +  sensitivity in the SLO1 BK channel conferred by the RCK1 domain. Nat Struct Mol Biol 15:403–410PubMedCrossRefGoogle Scholar
  17. 17.
    Schreiber M, Salkoff L (1997) A novel calcium-sensing domain in the BK channel. Biophys J 73:1355–1363PubMedCrossRefGoogle Scholar
  18. 18.
    Wei A, Solaro C, Lingle C, Salkoff L (1994) Calcium sensitivity of BK-type KCa channels determined by a separable domain. Neuron 13:671–681PubMedCrossRefGoogle Scholar
  19. 19.
    Bao L, Kaldany C, Holmstrand EC, Cox DH (2004) Mapping the BKCa channel’s “Ca2+ bowl”: side-chains essential for Ca2+ sensing. J Gen Physiol 123:475–489PubMedCrossRefGoogle Scholar
  20. 20.
    Bao L, Rapin AM, Holmstrand EC, Cox DH (2002) Elimination of the BK(Ca) channel’s high-affinity Ca2+ sensitivity. J Gen Physiol 120:173–189PubMedCrossRefGoogle Scholar
  21. 21.
    Kuo MMC, Haynes WJ, Loukin SH, Kung C, Saimi Y (2005) Prokaryotic K+ channels: From crystal structures to diversity. FEMS Microbiol Rev 29:961–985PubMedCrossRefGoogle Scholar
  22. 22.
    Kroning N, Willenborg M, Tholema N, Hanelt I, Schmid R, Bakker EP (2007) ATP binding to the KTN/RCK subunit KtrA from the K+-uptake system KtrAB of Vibrio alginolyticus: Its role in the formation of the KtrAB complex and its requirement in vivo. J Biol Chem 282:14018–14027PubMedCrossRefGoogle Scholar
  23. 23.
    Roosild TP, Castronovo S, Miller S, Li C, Rasmussen T, Bartlett W, Gunasekera B, Choe S, Booth IR (2009) KTN (RCK) domains regulate K+ channels and transporters by controlling the dimer-hinge conformation. Structure 17:893–903PubMedCrossRefGoogle Scholar
  24. 24.
    Zhang X, Solaro CR, Lingle CJ (2001) Allosteric regulation of BK channel gating by Ca2+ and Mg2+ through a nonselective, low affinity divalent cation site. J Gen Physiol 118:607–636PubMedCrossRefGoogle Scholar
  25. 25.
    Xia XM, Zeng X, Lingle CJ (2002) Multiple regulatory sites in large-conductance calcium-activated potassium channels. Nature 418:880–884PubMedCrossRefGoogle Scholar
  26. 26.
    Zeng XH, Xia XM, Lingle CJ (2005) Divalent cation sensitivity of BK channel activation supports the existence of three distinct binding sites. J Gen Physiol 125:273–286PubMedCrossRefGoogle Scholar
  27. 27.
    Zhang X, Zeng X, Lingle CJ (2006) Slo3 K+ channels: voltage and pH dependence of macroscopic currents. J Gen Physiol 128:317–336PubMedCrossRefGoogle Scholar
  28. 28.
    Yuan A, Santi CM, Wei A, Wang ZW, Pollak K, Nonet M, Kaczmarek L, Crowder CM, Salkoff L (2003) The sodium-activated potassium channel is encoded by a member of the Slo gene family. Neuron 37:765–773PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Frank J. Smith
    • 1
  • Brad S. Rothberg
    • 1
  1. 1.Department of BiochemistryTemple University School of MedicinePhiladelphiaUSA

Personalised recommendations