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Domain Structure and Conformational Changes in rat KV2.1 ion Channel

Journal of Neuroimmune Pharmacology volume 9, pages 727–739 (2014)Cite this article

Abstract

Voltage-gated potassium Kv2.1 channels are widely distributed in the central nervous system, specifically in neuroendocrine and endocrine cells. Their cytoplasmic C-termini are large and carry out many important functions. Here we provide the first direct structural evidence that each C-terminal part within the Kv2.1 ion channel is formed by two distinct domains (Kv2 and CTA). We expressed and purified two C-terminal truncation mutants of a rat Kv2.1 channel, lacking the entire C-termini or the CTA domain. Single particle electron microscopy was used to obtain three-dimensional reconstructions of purified C-terminal Kv2.1 mutants at 2.0 and 2.4 nm resolution. Comparison of these structures to each other and to the low-resolution EM structure of the full-length Kv2.1 channel revealed the exact locations of cytoplasmic Kv2 and CTA domains within the tetramer. Four Kv2 domains envelop the N-terminal T1 domain. The tetramer of the CTA domains underlies the Kv2-T1 complex and may also affect the channel’s surface expression. Subsequent molecular dynamics simulation and homology modeling produced open and closed structural models of the membrane part of the Kv2.1 channel.

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Abbreviations

Kv:

Voltage-gated potassium channel

AgTx2:

Agitoxin2

EM:

Electron microscopy

3D:

Three-dimensional

CTF:

Contrast transfer function

FSC:

Fourier shell correlation

MRA:

Multi-reference analysis

MSA:

Multivariate statistical analysis

MD:

Molecular dynamics

RMSD:

Root mean square deviation

VSD:

Voltage-sensor domain

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Acknowledgments

Authors thank Prof. C.Miller for the generous gift of the AgTx2 expression construct, Prof. B.Adair for providing the 3D EM map of the full-length Kv2.1 and Prof. M.Tarek for the model of Kv1.2 in closed state. We also like to thank Dr. A.Shaytan for help with MD experiments, E.Trifonova, C.Williams and K.Piasta for proof-reading the manuscript. The EM studies were performed at the User Facilities Center “Structural Diagnostics of Materials” at the A.V.Shoubnikov Institute of Crystallography RAS in Moscow, Russia. This work was supported by the FP7 program EDICT - EUROPEAN DRUG INITIATIVE ON CHANNELS AND TRANSPORTERS (#201924), and Federal programs of Russian ministry of education and science (#14.740.11.0255 and #14.740.11.0760). AG is supported by the Saint Petersburg State University project (#1.50.1038.2014). This article is dedicated to the memory of our good friend and colleague Dennis Wray.

Conflict of Interest

The authors declare no conflict of interests.

Author information

Affiliations

  1. Saint Petersburg State University, Saint Petersburg, 199034, Russia

    Anastasia Grizel

  2. N.A.Bakh Institute of Biochemistry RAS, Moscow, 119071, Russia

    Anna Popinako

  3. Faculty of Biology, M.V. Lomonosov Moscow University, 1 Leninskie Gory, Bld.12, 119991, Moscow, Russia

    Marina A. Kasimova, Maria Karlova, Mikhail M. Moisenovich & Olga S. Sokolova

  4. Institute of Membrane and Systems Biology, Leeds University, Leeds, LS2 9JT, UK

    Louisa Stevens

  5. A.V. Shoubnikov Institute of crystallography RAS, Moscow, 119333, Russia

    Olga S. Sokolova

Authors
  1. Anastasia Grizel
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  2. Anna Popinako
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  3. Marina A. Kasimova
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  4. Louisa Stevens
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  5. Maria Karlova
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  6. Mikhail M. Moisenovich
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  7. Olga S. Sokolova
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Corresponding author

Correspondence to Olga S. Sokolova.

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Fig. S1
figure 7

Single particle image processing of Kv2.1∆CTA and Kv2.1∆C. Gallery of filtered particle images of negatively stained Kv2.1∆C (a) and Kv2.1∆CTA (e). All particles are masked with the circular mask. Scale bar – 50 nm; Histograms displaying the classification of individual particles from the final round of refinement: (b) Kv2.1∆C, (f) Kv2.1∆CTA. Each spot represents an orientation of the two Euler angles: Phy and Theta; Eigen images, showing the 4-fold and 2-fold symmetry in the data sets, used for reconstruction of (c) Kv2.1∆C, and (g) Kv2.1∆CTA; 3D reconstructions of (d) Kv2.1∆C and (h) Kv2.1∆CTA before imposing the C4 symmetry, from left to right: top view, side view and bottom view. Scale bar – 10 nm. (GIF 809 kb)

Full size image
Fig. S2
figure 8

The resolution of the 3D reconstructions of Kv2.1∆CTA (dotted line) and Kv2.1∆C (solid line), according to the Fourier shell correlation, calculated between structures, each containing one half of the data. (GIF 23 kb)

Full size image
Fig. S3
figure 9

The amino acid sequence of the full-length Kv2.1 channel. The sequence, allocated to the T1 domain is marked with a bold font; the sequence, allocated to the membrane-embedded domain (MD) is marked with an underlined font; the location of Kv2 C-terminal domain is highlighted black; the location of the CTA domain is highlighted gray. The position of the S6-Kv2 linker is marked with gray symbols. (GIF 48 kb)

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High Resolution (TIFF 739 kb)

High Resolution (TIFF 15971 kb)

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Grizel, A., Popinako, A., Kasimova, M.A. et al. Domain Structure and Conformational Changes in rat KV2.1 ion Channel. J Neuroimmune Pharmacol 9, 727–739 (2014). https://doi.org/10.1007/s11481-014-9565-x

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  • DOI: https://doi.org/10.1007/s11481-014-9565-x

Keywords

  • Kv2 channel
  • Single particle electron microscopy
  • Cytoplasmic domains
  • Kv2
  • CTA
  • Molecular homology modeling
  • Conformational changes