1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics

Cooperative regulation by G proteins and Na+ of neuronal GIRK2 K+ channels

  1. Weiwei Wang
  2. Kouki K Touhara
  3. Keiko Weir
  4. Bruce P Bean
  5. Roderick MacKinnon  Is a corresponding author
  1. Howard Hughes Medical Institute, Rockefeller University, United States
https://doi.org/10.7554/eLife.15751
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  1. Weiwei Wang
  2. Kouki K Touhara
  3. Keiko Weir
  4. Bruce P Bean
  5. Roderick MacKinnon
(2016)
Cooperative regulation by G proteins and Na+ of neuronal GIRK2 K+ channels
eLife 5:e15751.
https://doi.org/10.7554/eLife.15751
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  3. Download .RIS

Abstract

G protein gated inward rectifier K+ (GIRK) channels open and thereby silence cellular electrical activity when inhibitory G protein coupled receptors (GPCRs) are stimulated. Here we describe an assay to measure neuronal GIRK2 activity as a function of membrane-anchored G protein concentration. Using this assay we show that four Gβγ subunits bind cooperatively to open GIRK2, and that intracellular Na+ - which enters neurons during action potentials - further amplifies opening mostly by increasing Gβγ affinity. A Na+ amplification function is characterized and used to estimate the concentration of Gβγ subunits that appear in the membrane of mouse dopamine neurons when GABAB receptors are stimulated. We conclude that GIRK2, through its dual responsiveness to Gβγ and Na+, mediates a form of neuronal inhibition that is amplifiable in the setting of excess electrical activity.

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Author details

  1. Weiwei Wang

    Laboratory of Molecular Neurobiology and Biophysics, Howard Hughes Medical Institute, Rockefeller University, New York, United States
    Competing interests
    The authors declare that no competing interests exist.

  2. Kouki K Touhara

    Laboratory of Molecular Neurobiology and Biophysics, Howard Hughes Medical Institute, Rockefeller University, New York, United States
    Competing interests
    The authors declare that no competing interests exist.

  3. Keiko Weir

    Laboratory of Molecular Neurobiology and Biophysics, Howard Hughes Medical Institute, Rockefeller University, New York, United States
    Competing interests
    The authors declare that no competing interests exist.

  4. Bruce P Bean

    Laboratory of Molecular Neurobiology and Biophysics, Howard Hughes Medical Institute, Rockefeller University, New York, United States
    Competing interests
    The authors declare that no competing interests exist.

  5. Roderick MacKinnon

    Laboratory of Molecular Neurobiology and Biophysics, Howard Hughes Medical Institute, Rockefeller University, New York, United States
    For correspondence
    mackinn@rockefeller.edu
    Competing interests
    The authors declare that no competing interests exist.

Reviewing Editor

  1. Kenton J Swartz, National Institutes of Health, United States

Ethics

Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to a protocol approved by the institutional animal care and use committee (IACUC) of Harvard Medical School (Protocol #02538-R98).

Version history

  1. Received: March 3, 2016
  2. Accepted: April 12, 2016
  3. Accepted Manuscript published: April 13, 2016 (version 1)
  4. Version of Record published: May 13, 2016 (version 2)

Copyright

© 2016, Wang et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Weiwei Wang
  2. Kouki K Touhara
  3. Keiko Weir
  4. Bruce P Bean
  5. Roderick MacKinnon
(2016)
Cooperative regulation by G proteins and Na+ of neuronal GIRK2 K+ channels
eLife 5:e15751.
https://doi.org/10.7554/eLife.15751

Share this article

https://doi.org/10.7554/eLife.15751

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Further reading

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    Kouki K Touhara, Weiwei Wang, Roderick MacKinnon
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    G protein gated inward rectifier potassium (GIRK) channels are gated by direct binding of G protein beta-gamma subunits (Gβγ), signaling lipids, and intracellular Na+. In cardiac pacemaker cells, hetero-tetramer GIRK1/4 channels and homo-tetramer GIRK4 channels play a central role in parasympathetic slowing of heart rate. It is known that the Na+ binding site of the GIRK1 subunit is defective, but the functional difference between GIRK1/4 hetero-tetramers and GIRK4 homo-tetramers remains unclear. Here, using purified proteins and the lipid bilayer system, we characterize Gβγ and Na+ regulation of GIRK1/4 hetero-tetramers and GIRK4 homo-tetramers. We find in GIRK4 homo-tetramers that Na+ binding increases Gβγ affinity and thereby increases the GIRK4 responsiveness to G protein stimulation. GIRK1/4 hetero-tetramers are not activated by Na+, but rather are in a permanent state of high responsiveness to Gβγ, suggesting that the GIRK1 subunit functions like a GIRK4 subunit with Na+ permanently bound.

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    Remodeling of skeletal muscle myosin metabolic states in hibernating mammals

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    Hibernation is a period of metabolic suppression utilized by many small and large mammal species to survive during winter periods. As the underlying cellular and molecular mechanisms remain incompletely understood, our study aimed to determine whether skeletal muscle myosin and its metabolic efficiency undergo alterations during hibernation to optimize energy utilization. We isolated muscle fibers from small hibernators, Ictidomys tridecemlineatus and Eliomys quercinus and larger hibernators, Ursus arctos and Ursus americanus. We then conducted loaded Mant-ATP chase experiments alongside X-ray diffraction to measure resting myosin dynamics and its ATP demand. In parallel, we performed multiple proteomics analyses. Our results showed a preservation of myosin structure in U. arctos and U. americanus during hibernation, whilst in I. tridecemlineatus and E. quercinus, changes in myosin metabolic states during torpor unexpectedly led to higher levels in energy expenditure of type II, fast-twitch muscle fibers at ambient lab temperatures (20 °C). Upon repeating loaded Mant-ATP chase experiments at 8 °C (near the body temperature of torpid animals), we found that myosin ATP consumption in type II muscle fibers was reduced by 77–107% during torpor compared to active periods. Additionally, we observed Myh2 hyper-phosphorylation during torpor in I. tridecemilineatus, which was predicted to stabilize the myosin molecule. This may act as a potential molecular mechanism mitigating myosin-associated increases in skeletal muscle energy expenditure during periods of torpor in response to cold exposure. Altogether, we demonstrate that resting myosin is altered in hibernating mammals, contributing to significant changes to the ATP consumption of skeletal muscle. Additionally, we observe that it is further altered in response to cold exposure and highlight myosin as a potentially contributor to skeletal muscle non-shivering thermogenesis.