Abstract
Ethanol consumption leads to a wide range of pharmacological effects by acting on the signaling proteins in the human nervous system, such as ion channels. Despite its familiarity and biological importance, very little is known about the molecular mechanisms underlying the ethanol action, due to extremely weak binding affinity and the dynamic nature of the ethanol interaction. In this research, we focused on the primary in vivo target of ethanol, G-protein-activated inwardly rectifying potassium channel (GIRK), which is responsible for the ethanol-induced analgesia. By utilizing solution NMR spectroscopy, we characterized the changes in the structure and dynamics of GIRK induced by ethanol binding. We demonstrated here that ethanol binds to GIRK with an apparent dissociation constant of 1.0 M and that the actual physiological binding site of ethanol is located on the cavity formed between the neighboring cytoplasmic regions of the GIRK tetramer. From the methyl-based NMR relaxation analyses, we revealed that ethanol activates GIRK by shifting the conformational equilibrium processes, which are responsible for the gating of GIRK, to stabilize an open conformation of the cytoplasmic ion gate. We suggest that the dynamic molecular mechanism of the ethanol-induced activation of GIRK represents a general model of the ethanol action on signaling proteins in the human nervous system.
| Original language | English |
|---|---|
| Pages (from-to) | 3858-3863 |
| Number of pages | 6 |
| Journal | Proceedings of the National Academy of Sciences of the United States of America |
| Volume | 115 |
| Issue number | 15 |
| DOIs | |
| Publication status | Published - 2018 |
Keywords
- Ethanol
- GIRK
- Ion channels
- NMR
ASJC Scopus subject areas
- General
Access to Document
Other files and links
Fingerprint
Dive into the research topics of 'Structural basis for the ethanol action on G-protein-activated inwardly rectifying potassium channel 1 revealed by NMR spectroscopy'. Together they form a unique fingerprint.Cite this
- APA
- Standard
- Harvard
- Vancouver
- Author
- BIBTEX
- RIS
In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 115, No. 15, 2018, p. 3858-3863.
Research output: Contribution to journal › Article › peer-review
}
TY - JOUR
T1 - Structural basis for the ethanol action on G-protein-activated inwardly rectifying potassium channel 1 revealed by NMR spectroscopy
AU - Toyama, Yuki
AU - Kano, Hanaho
AU - Mase, Yoko
AU - Yokogawa, Mariko
AU - Osawa, Masanori
AU - Shimada, Ichio
N1 - Funding Information: 27. Imai S, et al. (2012) Functional equilibrium of the KcsA structure revealed by NMR. J Biol Chem 287:39634–39641. Materials and Methods GIRKCP and the GIRK chimera proteins were expressed in Escherichia coli cells and purified by chromatography on Ni-NTA resin and size exclusion chromatography. All NMR measurements were performed on Bruker Avance 500, 600, or 800 spectrometers equipped with cryogenic probes. The MQ CPMG RD and methyl-HDR measurements of GIRKCP were performed using the reported pulse sequences (36, 37), and the dispersion curves and the ΔRMQ,ex values were simultaneously analyzed to obtain the exchange parameters (ΔωC, ΔωH, kex, and pE). Full experimental details can be found in SI Materials and Methods. ACKNOWLEDGMENTS. This work was supported in part by grants from the Japan New Energy and Industrial Technology Development Organization and the Ministry of Economy, Trade, and Industry (to I.S.); the Development of Core Technologies for Innovative Drug Development Based upon IT from Japan Agency for Medical Research and Development (to I.S.); the Ministry of Education, Culture, Sports, Science and Technology/Japan Society for the Promotion of Science KAKENHI Grants JP25121707 (to M.O.), JP16H01368 (to M.O.), JP17H03978 (to M.O.), and JP17H06097 (to I.S.); a grant from Takeda Science Foundation (to M.Y.); a grant from The Vehicle Racing Commemorative Foundation (to M.O.); and a grant from SENSHIN Medical Research Foundation (to M.O.). 28. Minato Y, et al. (2016) Conductance of P2X4 purinergic receptor is determined by conformational equilibrium in the transmembrane region. Proc Natl Acad Sci USA 113:4741–4746. 29. Osawa M, et al. (2009) Evidence for the direct interaction of spermine with the in-wardly rectifying potassium channel. J Biol Chem 284:26117–26126. 30. Yokogawa M, Osawa M, Takeuchi K, Mase Y, Shimada I (2011) NMR analyses of the Gβγ binding and conformational rearrangements of the cytoplasmic pore of G pro-tein-activated inwardly rectifying potassium channel 1 (GIRK1). J Biol Chem 286: 2215–2223. 31. Mase Y, Yokogawa M, Osawa M, Shimada I (2012) Structural basis for modulation of gating property of G protein-gated inwardly rectifying potassium ion channel (GIRK) by i/o-family G protein α subunit (Gαi/o). J Biol Chem 287:19537–19549. 32. Yokogawa M, Muramatsu T, Takeuchi K, Osawa M, Shimada I (2009) Backbone res-onance assignments for the cytoplasmic regions of G protein-activated inwardly rectifying potassium channel 1 (GIRK1). Biomol NMR Assign 3:125–128. 33. Tugarinov V, Hwang PM, Ollerenshaw JE, Kay LE (2003) Cross-correlated relaxation enhanced 1H–13C NMR spectroscopy of methyl groups in very high molecular weight proteins and protein complexes. J Am Chem Soc 125:10420–10428. 34. Kuo A, et al. (2003) Crystal structure of the potassium channel KirBac1.1 in the closed state. Science 300:1922–1926. 35. Enkvetchakul D, et al. (2004) Functional characterization of a prokaryotic Kir channel. J Biol Chem 279:47076–47080. 36. Toyama Y, Osawa M, Yokogawa M, Shimada I (2016) NMR method for characterizing microsecond-to-millisecond chemical exchanges utilizing differential multiple-quan-tum relaxation in high molecular weight proteins. J Am Chem Soc 138:2302–2311. 37. Korzhnev DM, Kloiber K, Kanelis V, Tugarinov V, Kay LE (2004) Probing slow dynamics in high molecular weight proteins by methyl-TROSY NMR spectroscopy: Application to a 723-residue enzyme. J Am Chem Soc 126:3964–3973. 38. Lesage F, et al. (1995) Molecular properties of neuronal G-protein-activated inwardly rectifying K+ channels. J Biol Chem 270:28660–28667. 39. Leal-Pinto E, et al. (2010) Gating of a G protein-sensitive mammalian Kir3.1 prokaryotic Kir channel chimera in planar lipid bilayers. J Biol Chem 285:39790–39800. 40. Chen L, et al. (2002) A glutamate residue at the C terminus regulates activity of in-ward rectifier K+ channels: Implication for Andersen’s syndrome. Proc Natl Acad Sci USA 99:8430–8435. 41. Meng XY, Zhang HX, Logothetis DE, Cui M (2012) The molecular mechanism by which PIP2 opens the intracellular G-loop gate of a Kir3.1 channel. Biophys J 102:2049–2059. 42. Li J, et al. (2016) Three pairs of weak interactions precisely regulate the G-loop gate of Kir2.1 channel. Proteins 84:1929–1937. 43. He C, et al. (2002) Identification of critical residues controlling G protein-gated in-wardly rectifying K+ channel activity through interactions with the β γ subunits of G proteins. J Biol Chem 277:6088–6096. 44. Kofuji P, Davidson N, Lester HA (1995) Evidence that neuronal G-protein-gated in-wardly rectifying K+ channels are activated by G β γ subunits and function as heter-omultimers. Proc Natl Acad Sci USA 92:6542–6546. 45. Goto NK, Gardner KH, Mueller GA, Willis RC, Kay LE (1999) A robust and cost-effective method for the production of Val, Leu, Ile (δ 1) methyl-protonated15N-, 13C-, 2H-la-beled proteins. J Biomol NMR 13:369–374. 46. Gans P, et al. (2010) Stereospecific isotopic labeling of methyl groups for NMR spectro-scopic studies of high-molecular-weight proteins. Angew Chem Int Ed Engl 49:1958–1962. 47. Ayala I, Sounier R, Usé N, Gans P, Boisbouvier J (2009) An efficient protocol for the complete incorporation of methyl-protonated alanine in perdeuterated protein. J Biomol NMR 43:111–119. 48. Wang C, Palmer AG, 3rd (2002) Differential multiple quantum relaxation caused by chemical exchange outside the fast exchange limit. J Biomol NMR 24:263–268. 49. Skrynnikov NR, Dahlquist FW, Kay LE (2002) Reconstructing NMR spectra of “in-visible” excited protein states using HSQC and HMQC experiments. J Am Chem Soc 124:12352–12360. BIOPHYSICS AND COMPUTATIONAL BIOLOGY Funding Information: ACKNOWLEDGMENTS. This work was supported in part by grants from the Japan New Energy and Industrial Technology Development Organization and the Ministry of Economy, Trade, and Industry (to I.S.); the Development of Core Technologies for Innovative Drug Development Based upon IT from Japan Agency for Medical Research and Development (to I.S.); the Ministry of Education, Culture, Sports, Science and Technology/Japan Society for the Promotion of Science KAKENHI Grants JP25121707 (to M.O.), JP16H01368 (to M.O.), JP17H03978 (to M.O.), and JP17H06097 (to I.S.); a grant from Takeda Science Foundation (to M.Y.); a grant from The Vehicle Racing Commemorative Foundation (to M.O.); and a grant from SENSHIN Medical Research Foundation (to M.O.). Funding Information: This work was supported in part by grants from the Japan New Energy and Industrial Technology Development Organization and the Ministry of Economy, Trade, and Industry (to I.S.); the Development of Core Technologies for Innovative Drug Development Based upon IT from Japan Agency for Medical Research and Development (to I.S.); the Ministry of Education, Culture, Sports, Science and Technology/Japan Society for the Promotion of Science KAKENHI Grants JP25121707 (to M.O.), JP16H01368 (to M.O.), JP17H03978 (to M.O.), and JP17H06097 (to I.S.); a grant from Takeda Science Foundation (to M.Y.); a grant from The Vehicle Racing Commemorative Foundation (to M.O.); and a grant from SENSHIN Medical Research Foundation (to M.O.). Publisher Copyright: © 2018 National Academy of Sciences. All rights reserved.
PY - 2018
Y1 - 2018
N2 - Ethanol consumption leads to a wide range of pharmacological effects by acting on the signaling proteins in the human nervous system, such as ion channels. Despite its familiarity and biological importance, very little is known about the molecular mechanisms underlying the ethanol action, due to extremely weak binding affinity and the dynamic nature of the ethanol interaction. In this research, we focused on the primary in vivo target of ethanol, G-protein-activated inwardly rectifying potassium channel (GIRK), which is responsible for the ethanol-induced analgesia. By utilizing solution NMR spectroscopy, we characterized the changes in the structure and dynamics of GIRK induced by ethanol binding. We demonstrated here that ethanol binds to GIRK with an apparent dissociation constant of 1.0 M and that the actual physiological binding site of ethanol is located on the cavity formed between the neighboring cytoplasmic regions of the GIRK tetramer. From the methyl-based NMR relaxation analyses, we revealed that ethanol activates GIRK by shifting the conformational equilibrium processes, which are responsible for the gating of GIRK, to stabilize an open conformation of the cytoplasmic ion gate. We suggest that the dynamic molecular mechanism of the ethanol-induced activation of GIRK represents a general model of the ethanol action on signaling proteins in the human nervous system.
AB - Ethanol consumption leads to a wide range of pharmacological effects by acting on the signaling proteins in the human nervous system, such as ion channels. Despite its familiarity and biological importance, very little is known about the molecular mechanisms underlying the ethanol action, due to extremely weak binding affinity and the dynamic nature of the ethanol interaction. In this research, we focused on the primary in vivo target of ethanol, G-protein-activated inwardly rectifying potassium channel (GIRK), which is responsible for the ethanol-induced analgesia. By utilizing solution NMR spectroscopy, we characterized the changes in the structure and dynamics of GIRK induced by ethanol binding. We demonstrated here that ethanol binds to GIRK with an apparent dissociation constant of 1.0 M and that the actual physiological binding site of ethanol is located on the cavity formed between the neighboring cytoplasmic regions of the GIRK tetramer. From the methyl-based NMR relaxation analyses, we revealed that ethanol activates GIRK by shifting the conformational equilibrium processes, which are responsible for the gating of GIRK, to stabilize an open conformation of the cytoplasmic ion gate. We suggest that the dynamic molecular mechanism of the ethanol-induced activation of GIRK represents a general model of the ethanol action on signaling proteins in the human nervous system.
KW - Ethanol
KW - GIRK
KW - Ion channels
KW - NMR
UR - http://www.scopus.com/inward/record.url?scp=85045199839&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85045199839&partnerID=8YFLogxK
U2 - 10.1073/pnas.1722257115
DO - 10.1073/pnas.1722257115
M3 - Article
C2 - 29581303
AN - SCOPUS:85045199839
SN - 0027-8424
VL - 115
SP - 3858
EP - 3863
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 15
ER -