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Crystal structure of an inactivated mutant mammalian voltage-gated K+ channel

Nature Structural & Molecular Biology volume 24, pages 857–865 (2017)Cite this article

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Abstract

C-type inactivation underlies important roles played by voltage-gated K+ (Kv) channels. Functional studies have provided strong evidence that a common underlying cause of this type of inactivation is an alteration near the extracellular end of the channel's ion-selectivity filter. Unlike N-type inactivation, which is known to reflect occlusion of the channel's intracellular end, the structural mechanism of C-type inactivation remains controversial and may have many detailed variations. Here we report that in voltage-gated Shaker K+ channels lacking N-type inactivation, a mutation enhancing inactivation disrupts the outermost K+ site in the selectivity filter. Furthermore, in a crystal structure of the Kv1.2-2.1 chimeric channel bearing the same mutation, the outermost K+ site, which is formed by eight carbonyl-oxygen atoms, appears to be slightly too small to readily accommodate a K+ ion and in fact exhibits little ion density; this structural finding is consistent with the functional hallmark of C-type inactivation.

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Figure 1: The V478W mutation enhances C-type inactivation in Shaker channels.
Figure 2: Structure and electron density map of the region around residue 406 in mutant molecule I.
Figure 3: Structure and electron density of the selectivity filter in mutant molecule I.
Figure 4: One-dimensional electron density profiles (electrons/Å3) sampled from Fo – Fc maps along the central axis of the filter of V406W-mutant molecule I.
Figure 5: Electron density profiles within the selectivity filter of V406W-mutant molecule I.
Figure 6: Structure and density of the V406W-mutant molecule II.
Figure 7: Crystal contacts between the turret region of the α subunit of molecule II and the β subunit of molecule I.
Figure 8: Electron density profiles within KcsA's selectivity filter.

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Acknowledgements

We thank P. De Weer and T. Hoshi (University of Pennsylvania) for critical review of our manuscript; S. Stayrook (University of Pennsylvania) for technical assistance with the in-house X-ray source; R. MacKinnon (Rockefeller University) for cDNA of the rat Kv1.2 α subunit in the pPIZC-C plasmid and C. Deutsch (University of Pennsylvania) for cDNA of the rat Kv β2 subunit in the PCR3A+ plasmid; S. Long (Memorial Sloan Kettering Cancer Center) for information on Kv expression and cryo-milling; the staff at beamlines 8.2.1 and 8.2.2, ALS, Lawrence Berkeley National Laboratory, and at beamline 23-IDB, APS, Argonne National Laboratory, for their support in data collection. Research reported in this publication was supported by the National Institute of Diabetes and Digestive and Kidney Diseases (DK109919) and the National Institute of General Medical Sciences (GM055560) of the National Institutes of Health to Z.L.

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  1. Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Victor Pau, Yufeng Zhou, Yajamana Ramu, Yanping Xu & Zhe Lu

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  1. Victor Pau
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All authors designed research, performed experiments and analyzed data. V.P., Y.Z., Y.R. and Z.L. wrote the manuscript.

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Integrated supplementary information

Supplementary Figure 1 Comparison of the tetrameric Shaker channel and a concatemer construct.

(a, b) Currents of tetrameric (a) and concatemer (b) channels, recorded from oocytes in the presence of 100 mM extracellular K+, as membrane voltage was stepped from the -80 mV holding potential to 80 mV in 10 mV increments and back to -80 mV. The dashed line identifies the zero current level. (c) Normalized tail currents (mean ± s.e.m.; n = 10 - 12) of tetrameric (open circles) and concatemer (open squares) channels plotted against membrane voltage; curves are fits of Boltzmann functions with midpoint voltage V1/2 (mean ± s.e.m.) of -30 ± 0.5 mV and apparent valence Z of 3.5 ± 0.3 for tetrameric channels or V1/2 of -30 ± 0.6 mV and apparent valence Z of 3.3 ± 0.2 for concatemer channels.

Supplementary Figure 2 Ionic currents of wild-type and mutant Kv1.2 channels, recorded from oocytes in the presence of 100 mM extracellular K+.

Ionic currents of wild-type (a) and mutant Kv1.2 (b) channels, The mutation in Kv1.2 corresponds to V478W in the Shaker channel. Currents were elicited by stepping membrane voltage from the -80 mV holding potential to 80 mV in 10 mV increments and back to -80 mV. The dashed line identifies the zero current level.

Supplementary Figure 3 Illustration of hydrogen bonds between the selectivity filter and the pore helix of Kv1.2-2.1, as defined by distances between 2.5 and 3.3 Å.

Carbon atoms of wild-type selectivity filter of two contiguous subunits (PDB: 2R9R) are shown as green and yellow sticks, oxygen atoms in red, and nitrogen atoms in blue; K+ ions and water molecules are shown as green and magenta spheres, respectively. Hydrogen bonds in the extracellular and intracellular regions are indicated by dashed blue and magenta lines, respectively.

Supplementary Figure 4 Comparison of Val377 positions in wild-type and V406W mutant I structures.

Section of super-positioned wild-type (orange) and mutant (blue) structures around the central axis of the selectivity filter, as viewed from the outside of the cell. The part of electron density (2Fo-Fc composite-omit map contoured at 1.5 σ) corresponding to Val 377 is shown.

Supplementary Figure 5 Comparison of Tyr373 in wild-type Kv1.2-2.1 and mutant I structures.

Structures of Gly372-Tyr373-Gly374 in wild-type (orange) and mutant (blue) channels are shown in two views (a and c versus b and d). The two structures were aligned according to Cα atoms of residues 364-372. The super-positioned Fo-Fc omit map of mutant molecule I, contoured at 5 σ (a, b) and 8 σ (c, d), was calculated with a model of the mutant molecule where Gly372-Tyr373-Gly374 were omitted.

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Pau, V., Zhou, Y., Ramu, Y. et al. Crystal structure of an inactivated mutant mammalian voltage-gated K+ channel. Nat Struct Mol Biol 24, 857–865 (2017). https://doi.org/10.1038/nsmb.3457

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