Evidence for the direct interaction of spermine with the inwardly retifying potassium channel

Masanori Osawa, Mariko Yokogawa, Takahiro Muramatsu, Yoko Mase, Ichio Shimada

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

The inwardly rectifying potassium channel (Kir) regulates resting membrane potential, K+ homeostasis, heart rate, and hormone secretion. The outward current is blocked in a voltage-dependent manner, upon the binding of intracellular polyamines or Mg2+ to the transmembrane pore domain. Meanwhile, electrophysiological studies have shown that mutations of several acidic residues in the intracellular regions affected the inward rectification. Although these acidic residues are assumed to bind polyamines, the functional role of the binding of polyamines and Mg2+ to the intracellular regions of Kirs remains unclear. Here, we report thermodynamic and structural studies of the interaction between polyamines and the cytoplasmic pore of mouse Kir3.1/GIRK1, which is gated by binding of G-protein βγ-subunit (Gβγ). ITC analyses showed that two spermine molecules bind to a tetramer of Kir3.1/GIRK1 with a dissociation constant of 26 μm, which is lower than other blockers. NMR analyses revealed that the spermine binding site is Asp-260 and its surrounding area. Small but significant chemical shift perturbations upon spermine binding were observed in the subunit-subunit interface of the tetramer, suggesting that spermine binding alters the relative orientations of the four subunits. Our ITC and NMR results postulated a spermine binding mode, where one spermine molecule bridges two Asp-260 side chains from adjacent subunits, with rearrangement of the subunit orientations. This suggests the functional roles of spermine binding to the cytoplasmic pore: stabilization of the resting state conformation of the channel, and instant translocation to the transmembrane pore upon activation through the Gβγ-induced conformational rearrangement.

Original languageEnglish
Pages (from-to)26117-26126
Number of pages10
JournalJournal of Biological Chemistry
Volume284
Issue number38
DOIs
Publication statusPublished - 2009 Sep 18
Externally publishedYes

Fingerprint

Spermine
Potassium Channels
Polyamines
Nuclear magnetic resonance
Inwardly Rectifying Potassium Channel
Molecules
Protein Subunits
Chemical shift
Thermodynamics
GTP-Binding Proteins
Membrane Potentials
Conformations
Carrier Proteins
Homeostasis
Stabilization
Heart Rate
Chemical activation
Binding Sites
Hormones
Membranes

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology
  • Medicine(all)

Cite this

Evidence for the direct interaction of spermine with the inwardly retifying potassium channel. / Osawa, Masanori; Yokogawa, Mariko; Muramatsu, Takahiro; Mase, Yoko; Shimada, Ichio.

In: Journal of Biological Chemistry, Vol. 284, No. 38, 18.09.2009, p. 26117-26126.

Research output: Contribution to journalArticle

@article{0fbaefd9dd6f47c0915c765bc9aaea15,
title = "Evidence for the direct interaction of spermine with the inwardly retifying potassium channel",
abstract = "The inwardly rectifying potassium channel (Kir) regulates resting membrane potential, K+ homeostasis, heart rate, and hormone secretion. The outward current is blocked in a voltage-dependent manner, upon the binding of intracellular polyamines or Mg2+ to the transmembrane pore domain. Meanwhile, electrophysiological studies have shown that mutations of several acidic residues in the intracellular regions affected the inward rectification. Although these acidic residues are assumed to bind polyamines, the functional role of the binding of polyamines and Mg2+ to the intracellular regions of Kirs remains unclear. Here, we report thermodynamic and structural studies of the interaction between polyamines and the cytoplasmic pore of mouse Kir3.1/GIRK1, which is gated by binding of G-protein βγ-subunit (Gβγ). ITC analyses showed that two spermine molecules bind to a tetramer of Kir3.1/GIRK1 with a dissociation constant of 26 μm, which is lower than other blockers. NMR analyses revealed that the spermine binding site is Asp-260 and its surrounding area. Small but significant chemical shift perturbations upon spermine binding were observed in the subunit-subunit interface of the tetramer, suggesting that spermine binding alters the relative orientations of the four subunits. Our ITC and NMR results postulated a spermine binding mode, where one spermine molecule bridges two Asp-260 side chains from adjacent subunits, with rearrangement of the subunit orientations. This suggests the functional roles of spermine binding to the cytoplasmic pore: stabilization of the resting state conformation of the channel, and instant translocation to the transmembrane pore upon activation through the Gβγ-induced conformational rearrangement.",
author = "Masanori Osawa and Mariko Yokogawa and Takahiro Muramatsu and Yoko Mase and Ichio Shimada",
year = "2009",
month = "9",
day = "18",
doi = "10.1074/jbc.M109.029355",
language = "English",
volume = "284",
pages = "26117--26126",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "38",

}

TY - JOUR

T1 - Evidence for the direct interaction of spermine with the inwardly retifying potassium channel

AU - Osawa, Masanori

AU - Yokogawa, Mariko

AU - Muramatsu, Takahiro

AU - Mase, Yoko

AU - Shimada, Ichio

PY - 2009/9/18

Y1 - 2009/9/18

N2 - The inwardly rectifying potassium channel (Kir) regulates resting membrane potential, K+ homeostasis, heart rate, and hormone secretion. The outward current is blocked in a voltage-dependent manner, upon the binding of intracellular polyamines or Mg2+ to the transmembrane pore domain. Meanwhile, electrophysiological studies have shown that mutations of several acidic residues in the intracellular regions affected the inward rectification. Although these acidic residues are assumed to bind polyamines, the functional role of the binding of polyamines and Mg2+ to the intracellular regions of Kirs remains unclear. Here, we report thermodynamic and structural studies of the interaction between polyamines and the cytoplasmic pore of mouse Kir3.1/GIRK1, which is gated by binding of G-protein βγ-subunit (Gβγ). ITC analyses showed that two spermine molecules bind to a tetramer of Kir3.1/GIRK1 with a dissociation constant of 26 μm, which is lower than other blockers. NMR analyses revealed that the spermine binding site is Asp-260 and its surrounding area. Small but significant chemical shift perturbations upon spermine binding were observed in the subunit-subunit interface of the tetramer, suggesting that spermine binding alters the relative orientations of the four subunits. Our ITC and NMR results postulated a spermine binding mode, where one spermine molecule bridges two Asp-260 side chains from adjacent subunits, with rearrangement of the subunit orientations. This suggests the functional roles of spermine binding to the cytoplasmic pore: stabilization of the resting state conformation of the channel, and instant translocation to the transmembrane pore upon activation through the Gβγ-induced conformational rearrangement.

AB - The inwardly rectifying potassium channel (Kir) regulates resting membrane potential, K+ homeostasis, heart rate, and hormone secretion. The outward current is blocked in a voltage-dependent manner, upon the binding of intracellular polyamines or Mg2+ to the transmembrane pore domain. Meanwhile, electrophysiological studies have shown that mutations of several acidic residues in the intracellular regions affected the inward rectification. Although these acidic residues are assumed to bind polyamines, the functional role of the binding of polyamines and Mg2+ to the intracellular regions of Kirs remains unclear. Here, we report thermodynamic and structural studies of the interaction between polyamines and the cytoplasmic pore of mouse Kir3.1/GIRK1, which is gated by binding of G-protein βγ-subunit (Gβγ). ITC analyses showed that two spermine molecules bind to a tetramer of Kir3.1/GIRK1 with a dissociation constant of 26 μm, which is lower than other blockers. NMR analyses revealed that the spermine binding site is Asp-260 and its surrounding area. Small but significant chemical shift perturbations upon spermine binding were observed in the subunit-subunit interface of the tetramer, suggesting that spermine binding alters the relative orientations of the four subunits. Our ITC and NMR results postulated a spermine binding mode, where one spermine molecule bridges two Asp-260 side chains from adjacent subunits, with rearrangement of the subunit orientations. This suggests the functional roles of spermine binding to the cytoplasmic pore: stabilization of the resting state conformation of the channel, and instant translocation to the transmembrane pore upon activation through the Gβγ-induced conformational rearrangement.

UR - http://www.scopus.com/inward/record.url?scp=70350023550&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=70350023550&partnerID=8YFLogxK

U2 - 10.1074/jbc.M109.029355

DO - 10.1074/jbc.M109.029355

M3 - Article

C2 - 19620244

AN - SCOPUS:70350023550

VL - 284

SP - 26117

EP - 26126

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 38

ER -