論文 - 横川　真梨子

NMR analyses of the Gbetagamma binding and conformational rearrangements of the cytoplasmic pore of G protein-activated inwardly rectifying potassium channel 1 (GIRK1)

Yokogawa, M.; Osawa, M.; Takeuchi, K.; Mase, Y.; Shimada, I.

J Biol Chem 286 （ 3 ） 2215 - 23 2011年01月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

G protein-activated inwardly rectifying potassium channel (GIRK) plays crucial roles in regulating heart rate and neuronal excitability in eukaryotic cells. GIRK is activated by the direct binding of heterotrimeric G protein betagamma subunits (Gbetagamma) upon stimulation of G protein-coupled receptors, such as M2 acetylcholine receptor. The binding of Gbetagamma to the cytoplasmic pore (CP) region of GIRK causes structural rearrangements, which are assumed to open the transmembrane ion gate. However, the crucial residues involved in the Gbetagamma binding and the structural mechanism of GIRK gating have not been fully elucidated. Here, we have characterized the interaction between the CP region of GIRK and Gbetagamma, by ITC and NMR. The ITC analyses indicated that four Gbetagamma molecules bind to a tetramer of the CP region of GIRK with a dissociation constant of 250 muM. The NMR analyses revealed that the Gbetagamma binding site spans two neighboring subunits of the GIRK tetramer, which causes conformational rearrangements between subunits. A possible binding mode and mechanism of GIRK gating are proposed.

NMR analyses of the interaction between CCR5 and its ligand using functional reconstitution of CCR5 in lipid bilayers

Yoshiura, C.; Kofuku, Y.; Ueda, T.; Mase, Y.; Yokogawa, M.; Osawa, M.; Terashima, Y.; Matsushima, K.; Shimada, I.

J Am Chem Soc 132 （ 19 ） 6768 - 77 2010年05月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

CC-chemokine receptor 5 (CCR5) belongs to the G protein-coupled receptor (GPCR) family and plays important roles in the inflammatory response. In addition, its ligands inhibit the HIV infection. Structural analyses of CCR5 have been hampered by its instability in the detergent-solubilized form. Here, CCR5 was reconstituted into high density lipoprotein (rHDL), which enabled CCR5 to maintain its functions for >24 h and to be suitable for structural analyses. By applying the methyl-directed transferred cross-saturation (TCS) method to the complex between rHDL-reconstituted CCR5 and its ligand MIP-1alpha, we demonstrated that valine 59 and valine 63 of MIP-1alpha are in close proximity to CCR5 in the complex. Furthermore, these results suggest that the protective influence on HIV-1 infection of a SNP of MIP-1alpha is due to its change of affinity for CCR5. This method will be useful for investigating the various and complex signaling mediated by GPCR, and will also provide structural information about the interactions of other GPCRs with lipids, ligands, G-proteins, and effector molecules.

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

Osawa, M.; Yokogawa, M.; Muramatsu, T.; Kimura, T.; Mase, Y.; Shimada, I.

J Biol Chem 284 （ 38 ） 26117 - 26 2009年09月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

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 betagamma-subunit (Gbetagamma). ITC analyses showed that two spermine molecules bind to a tetramer of Kir3.1/GIRK1 with a dissociation constant of 26 microM, 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 Gbetagamma-induced conformational rearrangement.

Backbone resonance assignments for the cytoplasmic regions of G protein-activated inwardly rectifying potassium channel 1 (GIRK1)

Yokogawa, M.; Muramatsu, T.; Takeuchi, K.; Osawa, M.; Shimada, I.

Biomol NMR Assign 3 （ 1 ） 125 - 8 2009年06月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

G protein-activated inwardly rectifying potassium channel (GIRK) plays crucial roles in regulating heart rate and neuronal excitability in eukaryotic cells. A variety of ligands, including heterotrimeric G protein betagamma subunits (G betagamma), bind to the cytoplasmic regions of GIRK and modulate its activity. We established the backbone resonance assignments of (2)H/(13)C/(15)N-labeled cytoplasmic regions of mouse GIRK1, which form a tetramer with a molecular weight of 96 K.

Bead-linked proteoliposomes: a reconstitution method for nmr analyses of membrane protein-ligand interactions

Yokogawa, M.; Takeuchi, K.; Shimada, I.

J Am Chem Soc 127 （ 34 ） 12021 - 7 2005年08月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

Structural information about the interactions between membrane proteins and their ligands provides insights into the membrane protein functions. A variety of surfactants have been used for structural analyses of membrane proteins, and in some cases, they yielded successful results. However, the use of surfactants frequently increases the conformational instability of membrane proteins and distorts their normal function. Here, we propose a new strategy of membrane protein reconstitution into lipid bilayers on affinity beads, which maintains the native conformation and function of the protein for NMR studies. The reconstituted membrane proteins are suitable for NMR analyses of interactions, by using the transferred cross-saturation method. The strategy was successfully applied to the interaction between a potassium ion channel, KcsA, and a pore-blocker, agitoxin2 (AgTx2). This strategy would be useful for analyzing the interactions between various membrane proteins and their ligands.

Structural basis of the KcsA K(+) channel and agitoxin2 pore-blocking toxin interaction by using the transferred cross-saturation method

Takeuchi, K.; Yokogawa, M.; Matsuda, T.; Sugai, M.; Kawano, S.; Kohno, T.; Nakamura, H.; Takahashi, H.; Shimada, I.

Structure 11 （ 11 ） 1381 - 92 2003年11月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

We have determined the binding site on agitoxin2 (AgTx2) to the KcsA K(+) channel by a transferred cross-saturation (TCS) experiment. The residues significantly affected in the TCS experiments formed a contiguous surface on AgTx2, and substitutions of the surface residues decreased the binding affinity to the KcsA K(+) channel. Based on properties of the AgTx2 binding site with the KcsA K(+) channel, we present a surface motif that is observed in pore-blocking toxins affecting the K(+) channel. Furthermore, we also explain the structural basis of the specificity of the K(+) channel to the toxins. The TCS method utilized here is applicable not only for the channels, which are complexed with other inhibitors, but also with a variety of regulatory molecules, and provides important information about their interface in solution.