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Structural basis of PIP2 activation of the classical inward rectifier K+ channel Kir2.2

Abstract

The regulation of ion channel activity by specific lipid molecules is widely recognized as an integral component of electrical signalling in cells1,2. In particular, phosphatidylinositol 4,5-bisphosphate (PIP2), a minor yet dynamic phospholipid component of cell membranes, is known to regulate many different ion channels2,3,4,5,6,7,8. PIP2 is the primary agonist for classical inward rectifier (Kir2) channels, through which this lipid can regulate a cell’s resting membrane potential2,7,8,9. However, the molecular mechanism by which PIP2 exerts its action is unknown. Here we present the X-ray crystal structure of a Kir2.2 channel in complex with a short-chain (dioctanoyl) derivative of PIP2. We found that PIP2 binds at an interface between the transmembrane domain (TMD) and the cytoplasmic domain (CTD). The PIP2-binding site consists of a conserved non-specific phospholipid-binding region in the TMD and a specific phosphatidylinositol-binding region in the CTD. On PIP2 binding, a flexible expansion linker contracts to a compact helical structure, the CTD translates 6 Å and becomes tethered to the TMD and the inner helix gate begins to open. In contrast, the small anionic lipid dioctanoyl glycerol pyrophosphatidic acid (PPA) also binds to the non-specific TMD region, but not to the specific phosphatidylinositol region, and thus fails to engage the CTD or open the channel. Our results show how PIP2 can control the resting membrane potential through a specific ion-channel-receptor–ligand interaction that brings about a large conformational change, analogous to neurotransmitter activation of ion channels at synapses.

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Figure 1: Effect of a short-chain PIP 2 on Kir2.2.
Figure 2: PIP 2 -binding site.
Figure 3: Conserved non-specific lipid-binding site in Kir channels.
Figure 4: A proposed mechanism of Kir2.2 activation by PIP2.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the reported crystal structures have been deposited into the Protein Data Bank under accession codes 3SPI (wild-type PIP2), 3SPC (wild-type PPA), 3SPH (PIP2(I223L)), 3SPJ (apo(I223L)) and 3SPG (PIP2(R186A)).

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Acknowledgements

We thank staff members at NSLS X29 and X25, Brookhaven National Laboratory for beamline assistance, members of the Gadsby laboratory (Rockefeller University) for help in Xenopus oocyte preparation, R. Molday (University of British Columbia) for providing the anti-1D4 tag cell line and members of the MacKinnon laboratory for helpful suggestions. R.M. is an investigator in the Howard Hughes Medical Institute.

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Authors

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S.B.H. purified and crystallized the protein; collected, processed and refined crystallographic data, and performed electrophysiology experiments. X.T. aided in experimental design and provided assistance in all aspects of the project. R.M. designed the study and analysed data. All authors wrote and discussed the manuscript.

Corresponding author

Correspondence to Roderick MacKinnon.

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The authors declare no competing financial interests.

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Hansen, S., Tao, X. & MacKinnon, R. Structural basis of PIP2 activation of the classical inward rectifier K+ channel Kir2.2. Nature 477, 495–498 (2011). https://doi.org/10.1038/nature10370

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