X-ray structure of the mammalian GIRK2–βγ G-protein complex

Abstract

G-protein-gated inward rectifier K+ (GIRK) channels allow neurotransmitters, through G-protein-coupled receptor stimulation, to control cellular electrical excitability. In cardiac and neuronal cells this control regulates heart rate and neural circuit activity, respectively. Here we present the 3.5 Å resolution crystal structure of the mammalian GIRK2 channel in complex with βγ G-protein subunits, the central signalling complex that links G-protein-coupled receptor stimulation to K+ channel activity. Short-range atomic and long-range electrostatic interactions stabilize four βγ G-protein subunits at the interfaces between four K+ channel subunits, inducing a pre-open state of the channel. The pre-open state exhibits a conformation that is intermediate between the closed conformation and the open conformation of the constitutively active mutant. The resultant structural picture is compatible with ‘membrane delimited’ activation of GIRK channels by G proteins and the characteristic burst kinetics of channel gating. The structures also permit a conceptual understanding of how the signalling lipid phosphatidylinositol-4,5-bisphosphate (PIP2) and intracellular Na+ ions participate in multi-ligand regulation of GIRK channels.

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Figure 1: Functional properties of the channel.
Figure 2: Overall structure of the GIRK–Gβγ complex.
Figure 3: The GIRK–Gβγ binding interface.
Figure 4: The role of electrostatics at the GIRK–Gβγ interface.
Figure 5: Gβγ-induced conformational changes in GIRK.
Figure 6: A model of gating regulation of GIRK channels.

Accession codes

Accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the reported crystal structure have been deposited into the Protein Data Bank under accession code 4KFM.

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Acknowledgements

We thank P. Hoff and members of D. Gadsby’s laboratory (Rockefeller University) for assistance with oocyte preparation; Y. Hsiung for assistance with insect cell culture; R. Sanishvili, N. Venugopalan, and S. Corcoran (GM/CA, Advanced Photon Source, Argonne National laboratory) for assistance at the synchrotron; and members of the MacKinnon laboratory. The use of the Rigaku/MSC microMax 007HF and Formulator robot in the Rockefeller University Structural Biology Resource Center was made possible by Grant Numbers 1S10RR022321-01 and 1S10RR027037-01, respectively, from the National Center for Research Resources of the National Institutes of Health (NIH). R.M. is an investigator in the Howard Hughes Medical Institute.

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Authors

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M.R.W. performed the experiments. M.R.W and R.M. analysed the data and wrote the paper.

Corresponding author

Correspondence to Roderick MacKinnon.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Table 1, Supplementary Figures 1-6, Supplementary Video Legends 1-3 and additional references. (PDF 4743 kb)

Effect of Gβγ binding on the GIRK channel

A morph between the GIRK-PIP2 structure (PDB ID: 3SYA) (shown first) and the GIRK-PIP2-Gβγ structure (shown second). The structures are aligned by a conformationally inert region around the selectivity filter at the top of the transmembrane domain. At 10s, a top-down view is shown. At 20s, a closeup of the inner helix gate is shown. (MOV 21853 kb)

Hypothesized complete gating mechanism

A morph between the GIRK-PIP2 structure (PDB ID: 3SYA) (shown first), the GIRK-PIP2-Gβγ structure (shown second), and the GIRK(R201A)-PIP2 structure (PDB ID: 3SYQ) (shown third). All of the structures are aligned by a conformationally inert region around the selectivity filter at the top of the transmembrane domain. At 20s, a top-down view is shown. (MOV 31295 kb)

Effect of Gβγ binding on the GIRK channel cytoplasmic domain, independent of the rigid body rotation

GIRK-PIP2-Gβγ structure (shown second). The structures are aligned by the cytoplasmic domain to show the conformational changes that happen in the cytoplasmic domain independent of the rigid-body rotation highlighted in Supplementary Videos 1 and 2. The video starts with a close-up view of the Gβγ binding site on GIRK, then starts zooming out at 10s to show the whole cytoplasmic domain. (MOV 17385 kb)

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Whorton, M., MacKinnon, R. X-ray structure of the mammalian GIRK2–βγ G-protein complex. Nature 498, 190–197 (2013). https://doi.org/10.1038/nature12241

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