Abstract
Small-conductance Ca2+-activated K+ channels (SK channels)1,2 are independent of voltage and gated solely by intracellular Ca2+. These membrane channels are heteromeric complexes that comprise pore-forming α-subunits and the Ca2+-binding protein calmodulin (CaM). CaM binds to the SK channel through the CaM-binding domain (CaMBD), which is located in an intracellular region of the α-subunit immediately carboxy-terminal to the pore3,4. Channel opening is triggered when Ca2+ binds the EF hands in the N-lobe of CaM4. Here we report the 1.60 Å crystal structure of the SK channel CaMBD/Ca2+/CaM complex. The CaMBD forms an elongated dimer with a CaM molecule bound at each end; each CaM wraps around three α-helices, two from one CaMBD subunit and one from the other. As only the CaM N-lobe has bound Ca2+, the structure provides a view of both calcium-dependent and -independent CaM/protein interactions. Together with biochemical data, the structure suggests a possible gating mechanism for the SK channel.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Köhler, M. et al. Small-conductance, calcium-activated potassium channels from mammalian brain. Science 273, 1709–1714 (1998).
Vergara, C., Lattore, R., Marrion, N. V. & Adelman, J. P. Calcium-activated potassium channels. Curr. Opin. Neurobiol. 8, 321–329 (1998).
Xia, X.-M. et al. Mechanism of calcium gating in small-conductance calcium-activated potassium channels. Nature 395, 503–507 (1998).
Keen, J. E. et al. Domains responsible for constitutive and Ca2+-dependent interactions between calmodulin and small conductance Ca2+-activated potassium channels. J. Neurosci. 19, 8830–8838 (2000).
Ikura, M. et al. Solution structure of a calmodulin-target peptide complex by multidimensional NMR. Science 256, 632–638 (1992).
Ikura, M., Barbato, G., Klee, C. B. & Bax, A. Solution structure of calmodulin and its complex with a myosin light chain kinase fragment. Cell Calcium 13, 391–400 (1992).
Meador, W. E., Means, A. R. & Quiocho, F. A. Target enzyme recognition by calmodulin: 2.4 Å structure of a calmodulin-peptide complex. Science 257, 1251–1255 (1992).
Osava, M. et al. A novel target recognition revealed by calmodulin in complex with Ca2+ calmodulin dependent kinase kinase. Nature Struct. Biol. 6, 819–826 (1999).
Elhorst, B. et al. NMR solution structure of a complex of calmodulin with a binding peptide of the Ca2+ pump. Biochemistry 38, 12320–12326 (1999).
Rhoads, A. R. & Friedberg, F. Sequence motifs for calmodulin binding. FASEB J. 11, 331–339 (1997).
Babu, Y. S. et al. Three-dimensional structure of calmodulin. Nature 315, 37–40 (1985).
Chattopadhyaya, R., Meador, W. E., Means, A. R. & Quiocho, F. A. Calmodulin structure refined at 1. 7 Å resolution. J. Mol. Biol. 228, 1177–1192 (1992).
Zhang, M., Tanaka, T. & Ikura, M. Calcium-induced conformational transition revealed by the solution structure of the apo calmodulin. Nature Struct. Biol. 2, 758–767 (1995).
Kuboniwa, H. et al. Solution structure of calcium-free calmodulin. Nature Struct. Biol. 2, 768–776 (1995).
Finn, B. E. et al. Calcium-induced structural changes and domain autonomy in calmodulin. Nature Struct. Biol. 2, 777–783 (1995).
Liu, Y., Holgrem, M., Jurman, M. E. & Yellen, G. Gated access to the pore of a voltage-dependent K+ channel. Neuron 19, 175–184 (1997).
del Camino, D., Holgrem, M., Liu, Y. & Yellen, G. Blocker protection in the pore of a voltage-gated K+ channel and its structural implications. Nature 403, 321–325 (2000).
Holmgren, M., Shin, K. S. & Yellen, G. The activation gate of a voltage-gated K+ channel can be trapped in the open state by an intersubunit metal bridge. Neuron 21, 617–621 (1998).
Doyle, D. A. et al. The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science 280, 69–77 (1998).
Perozo, E., Cortes, D. M. & Cuello, L. G. Structural rearrangements underlying K+-channel activation gating. Science 285, 73–78 (1999).
Fragioni, J. V. & Neel, B. G. Solubilization and purification of enzymatically active glutatione S-transferase (pGEX) fusion proteins. Anal. Biochem. 210, 179–187 (1993).
Howard, A. J., Nielson, C. & Xuong, N. H. Software for a diffractometer with multiwire area detector. Methods Enzymol. 114, 452–472 (1985).
Furey, W. B. & Swaminathan, S. PHASE-95: a program package for the processing and analysis of diffraction data from macromolecules. Methods Enzymol. 277, 590–620 (1997).
Jones, T. A., Zou, J.-Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991).
Tronrud, D. E., TenEyck, L. F. & Matthews, B. W. An efficient general purpose least-squares refinement program for macromolecular structures. Acta Crystallogr. A 43, 489–501 (1985).
Kissinger, C. R., Gehlaer, D. K. & Fogel, D. B. Rapid automated molecular replacement by evolutionary search. Acta Crystallogr. D 55, 484–491 (1999).
Brünger, A. T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998).
Laskowski, R. A., MacArthur, M. W. & Thorton, J. M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–291 (1993).
Kraulis, P. J. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24, 946–950 (1991).
Nicholls, A., Sharp, K. & Honig, B. H. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins Struct. Funct. Genet. 11, 281–296 (1991).
Acknowledgements
M.A.S. is a Burroughs Wellcome Career Development Awardee. This work was supported by grants from the NIH, the Muscular Dystrophy Association, and the Human Frontiers of Science Foundation. Intensity data collected at the Stanford Synchrotron Radiation Laboratory (SSRL) was carried out under the auspices of the SSRL biotechnology program, which is supported by the National Institutes of Health, National Center for Research Resources, Biomedical Technology Program, and by the Department of Energy, Office of Biological and Environmental Research.
Author information
Authors and Affiliations
Corresponding author
Supplementary information
1. Rat calmodulin was cloned into pET23b, expressed in BL-21(DE3), and purified on a low substitution phenyl sepharose column (Pharmacia). The codons of the rSK2 CaMBD were optimized for expression in E. coli and the synthetic gene cloned into pET33b. The C-terminal histidine fusion was expressed, solubilized with sarkosyl21, and purified on a nickel column. The 1:1 protein complex was formed by slowly adding the CaMBD to CaM. All purified protein concentrations were determined using predicted extinction coefficients.
The sequence of the CaMBD (including the His-tag) used for expression is:
MGRKLELTKAEKHVHNFMMDTQLTKRVKNAAANVLRETWLIYKNTKLVKKIDHAKVRKHQRKFLQAIHQLRSVKMEQRKLNDQANTLVDLAKTQLEHHHHHH.
This includes the predicted last ~8 residues of S6 and the region connecting S6 to the CaMBD. This does not include residues 491 to 580, with unknown function, that forms the very C-terminal part of the channel following the CaMBD.
2. DLS was carried out using a 2001 DynaPro Dynamic Light Scattering Instrument. All solutions were filtered through 0.1 m m Anotop filters (Whatman) to remove particulates. Duplicate runs were made taking 20 to 40 scans at 30 s each at 22° C and analyzed by the DYNAMICS software, version 3.30. The CaMBD-CaM-Ca2+ complex was highly monodisperse and could only be modelled using a monomodal fit, giving a molecular weight of 61 +/- 3 kDa, concordant with a dimeric complex. This data analysis gave a baseline error of 1.001 and a relative polydispersity of 20% (baseline errors <1.005 and relative polydispersities < 25% indicate monodisperse species). The complex without Ca2+ was slightly aggregated (baseline error = 1.006, relative polydispersity = 28%) and the data were fit with a bimodal analysis resulting in a population in which 78% of the species had a molecular weight of 30 +/- 6 kDa, consistent with a monomer, and 22% of the population had a molecular weight of 541 kDa (aggregated). Notably, addition of Ca2+ (2 mM) to this Ca2+-free complex resulted in the formation of a cleanly monodisperse solution with a molecular weight of 62 +/- 4 kDa.
Sedimentation equilibrium measurements were performed on a Beckman model E analytical ultracentrifuge. Absorbance was measured at 285 nm. Runs were carried out in an AnF-Ti rotor using 12 mm filled Epon double-sector centerpieces. The Ca2+-free complex was measured at a concentration of ~3 mg/ml in Buffer A (50 mM NaCl, 10 mM Tris, pH 7.5, 5mM EGTA) at 20,000 rpm while the Ca2+-bound complex was measured at a concentration of ~3 mg/ml in Buffer B (50 mM NaCl, 10 mM Tris, pH=7.5, 10 mM CaCl2) at 13,000 rpm. Buffer A (or B) was placed in the reference compartment, and sample solutions were loaded into sample compartments of double sector cells. The partial specific volume, v2, was calculated to 0.73 ml/g based on the amino acid composition and the consensus scale of Perkins. The minimum molecular mass was derived from the slope of In c versus r2 plots (r = distance from the rotor center) after linearity has been optimized by adjusting the baseline. These measurements gave molecular weights of 29 +/- 5 kDa and 54 +/- 5 kDa for the Ca2+-free complex and the Ca2+-bound complex, respectively.
Figure 1.
(GIF 35 KB)
Schematic diagram of CaMBD-CaM interactions. Shown are contacts <4.1 Å. In this figure the CaMBD is represented as helical cylinders and contacts from CaM residues are shown in colour and indicated by arrows. Water-mediated contacts between CaMBD and CaM residues are also shown with the bridging water molecule indicated in parentheses by number (ie. S5). The top schematic details the Ca2+-independent C-lobe CaM interactions to the CaMBD. CaM residues mediating these contacts are coloured green as are the arrows that point from each CaM residue to the CaMBD residues that are contacted. The CaM N-lobe makes contacts to both CaMBD subunits in the dimer. These contacts are shown in the middle and bottom schematic. Not shown (for space reasons) are contacts between CaMBD residues Phe456 to CaM residues Ile85. Also not shown are long range electrostatic contacts between CaMBD residue Lys467 and CaM acidic residues Glu83, Glu84 and Glu87.
Figure 2.
(JPG 48.8 KB)
Composite simulated annealing omit map (blue mesh) of the repacked C-lobe region superimposed on the final refined model (red and yellow sticks). The map, contoured at 1.5 s, was calculated following simulated annealing at a starting temperature of 5000 K (calculated for uFo - vFc where u = 2 and v = 1 giving a 2Fo - Fc map27. The map was "constructed" following omission of 5% regions of the model and subsequently combined to give an omit map for the entire molecule).
Table 1
(GIF 27.3 KB)
CaMBD-CaM-Ca2+ Structure Determination and Refinement
*Rsym =SS∑|Ihkl-Ihkl(j)|/ ∑NIhkl, where Ihkl(j) is observed intensity and Ihkl is the final average value of intensity.
†Phasing power = rms(|Fh|/E), |Fh| = heavy atom structure factor amplitude and E = residual lack of closure error.
‡RCullis = S||Fh(obs)|-|Fh(calc)||/S|Fh(obs)| for centric reflections where |Fh(obs)| = observed heavy atom structure factor amplitudes and |Fh(calc)| = calculated heavy atom structure factor amplitude.
§Riso = S|| Fph| - | Fp||/S|Fp|, where |Fp| is the protein structure factor amplitude and |Fph| is the heavy atom derivative structure factor amplitude.
||Figure of Merit = <|SP(a)eia/SP(a)|>, where a is the phase and P(a) is the phase probability distribution.
¶Rcryst = S||Fobs| - |Fcalc||/S|Fobs| and Rfree = S||Fobs| - |Fcalc||/S|Fobs|; where all reflections belong to a test set of 10% data randomly selected in CNS.
Rights and permissions
About this article
Cite this article
Schumacher, M., Rivard, A., Bächinger, H. et al. Structure of the gating domain of a Ca2+-activated K+ channel complexed with Ca2+/calmodulin. Nature 410, 1120–1124 (2001). https://doi.org/10.1038/35074145
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1038/35074145
This article is cited by
-
Bioinspired nanofluidic iontronics for brain-like computing
Nano Research (2024)
-
Overexpression of the elongation factor MtEF1A1 promotes salt stress tolerance in Arabidopsis thaliana and Medicago truncatula
BMC Plant Biology (2023)
-
Age-related hearing loss pertaining to potassium ion channels in the cochlea and auditory pathway
Pflügers Archiv - European Journal of Physiology (2021)
-
Calmodulin7: recent insights into emerging roles in plant development and stress
Plant Molecular Biology (2021)
-
Critical regulation of atherosclerosis by the KCa3.1 channel and the retargeting of this therapeutic target in in-stent neoatherosclerosis
Journal of Molecular Medicine (2019)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.