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Structure prediction for the down state of a potassium channel voltage sensor

Abstract

Voltage-gated potassium (Kv) channels, essential for regulating potassium uptake and cell volume in plants and electrical excitability in animals, switch between conducting and non-conducting states as a result of conformational changes in the four voltage-sensing domains (VSDs) that surround the channel pore1. This process, known as gating, is initiated by a cluster of positively charged residues on the fourth transmembrane segment (S4) of each VSD, which drives the VSD into a ‘down state’ at negative voltages and an ‘up state’ at more positive voltages2. The crystal structure of Kv1.2 probably corresponds to the up state3, but the local environment of S4 in the down state and its motion in voltage gating remains unresolved4,5,6. Here we employed several conditional lethal/second-site suppressor yeast screens to determine the transmembrane packing of the VSD in the down state. This screen relies on the ability of KAT1, a eukaryotic Kv channel, to conduct potassium when its VSDs are in the down state, thereby rescuing potassium-transport-deficient yeast7. Starting with KAT1 channels bearing conditional lethal mutations, we identified second-site suppressor mutations throughout the VSD that recover yeast growth. We then constructed a down state model of the channel using six pairs of interacting residues as structural constraints and verified this model by engineering suppressor mutations on the basis of spatial considerations. A comparison of this down state model with the up state Kv1.2 structure suggests that the VSDs undergo large rearrangements during gating, whereas the S4 segment remains positioned between the central pore and the remainder of the VSD in both states.

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Figure 1: Identified conditional lethal and second-site suppressor pairs.
Figure 2: A single model is consistent with the experimental data.
Figure 3: Verification of the model.
Figure 4: The extent of S4 movement during voltage gating.

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Acknowledgements

We thank J. Schroeder for the KAT1 construct; S. Kurtz for providing the yeast strain; D. Minor for experimental guidance; W. Zhou for experimental assistance; A. Fay, F. Haass and S. Nayak for critically reading the manuscript; and members of the Jan Laboratory and B. Tu for their support, advice and input at all stages of this project. This work was supported by a National Science Foundation Interdisciplinary Informatics Fellowship (M.G.), an American Heart Association Pre-doctoral Fellowship (H.C.L.), an NIH Structural Biology Training grant (H.C.L.), and an R01 grant from the NIMH. Y.N.J. and L.Y.J. are HHMI investigators.

Author Contributions M.G. carried out all the computational aspects of this project and H.C.L. performed the yeast screens assisted by M.J. The project was a collaborative effort through iterative cycles of experiment and computational model building. All authors discussed the results and commented on the manuscript.

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Correspondence to Lily Yeh Jan.

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Supplementary information

Supplementary Information

This file contains Supplementary Discussion, Supplementary Methods, Supplementary Figures 1-4, Supplementary Table 1-2, Supplementary Movie legend and additional references. (PDF 448 kb)

Supplementary Video 1

This file contains Supplementary Movie which shows the down and up gating states. (MOV 3605 kb)

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Grabe, M., Lai, H., Jain, M. et al. Structure prediction for the down state of a potassium channel voltage sensor. Nature 445, 550–553 (2007). https://doi.org/10.1038/nature05494

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