GPCR Activation of the G Protein-Gated Inward-Rectifier Potassium Channel

Heart rate is tightly regulated by the combined effects of the sympathetic and parasympathetic branches of the autonomic nervous system. These two branches control heart rate by stimulating different G protein-coupled receptors (GPCRs), which in turn activate ion channels that modify the electrical properties of cardiac pacemaker cells. Sympathetic stimulation accelerates heart rate through activation of beta-adrenergic receptors (βARs), which open excitatory ion channels through the stimulatory G protein (Gαs) pathway. Parasympathetic stimulation slows heart rate through activation of the muscarinic acetylcholine receptor M2 (M2Rs), which inhibits the effect of sympathetic stimulation through the inhibitory G protein (Gαi) pathway. M2Rs also release G protein beta-gamma subunits (Gβγ), which slow heart rate by activating G protein-gated inwardrectifier potassium (GIRK) channels. Interestingly, βARs also release the very same free Gβγ, but GIRK is not activated. The molecular mechanism underlying this specificity is poorly characterized. What is the molecular basis behind signaling specificity? It has been proposed that GIRK channels form a macromolecular supercomplex with Gαi-coupled receptors and G proteins, allowing released Gβγ to bind to and activate GIRK by proximity. However the evidence for the existence of the complex remains controversial. In the first part of my thesis, I challenge the supercomplex hypothesis by providing three experimental sets against the theory. First, GIRK co-localization with GPCRs shows no preference for M2Rs over β2ARs. Second, β2ARs do not activate GIRK channels even when they are co-localized. Third, neither Gαi1 nor G protein heterotrimers functionally interact with purified GIRK1/4 channels in the planar lipid bilayer system. I conclude that protein co-localization is not the underlying mechanism to explain why GIRK channels are specifically activated by Gαi-coupled receptors. I then set out to determine the molecular basis behind signaling specificity. Using electrophysiological technologies and bioluminescent resonance electron transfer (BRET) assays, I show that M2Rs catalyze release of Gβγ subunits at higher rates than β2ARs, generating higher Gβγ concentrations that activate GIRK and regulate other targets of Gβγ. The higher rate of Gβγ release is attributable to a faster GPCR-G protein association rate in M2Rs compared to β2ARs. I conclude that the activity of GIRK channels is simply determined by the efficiency of Gβγ release from GPCRs. Physiologically, only Gαi-coupled receptors can provide sufficient concentration of Gβγ to activate GIRK channels. In the second part of my thesis, I present my work on the functional characterization of Gβγ and Na+ regulation of two cardiac GIRK channels, GIRK1/4 hetero-tetramers and GIRK4 homo-tetramers. It is known that cardiac GIRK channels are composed of GIRK1/4 heterotetramers and GIRK4 homo-tetramers. However little is known about the functional difference between the two channels. Using purified proteins and the planar lipid bilayer system, I find that Na+ binding increases Gβγ affinity in GIRK4 homo-tetramers 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

ELECTROCHEMICAL PROPERTIES OF MUIJTIWALL CARBON NANOTUBES

Article先進繊維技術科学に関する研究報告 平成11年度成果報告 6: 17-19(2000)research repor

Structural Basis for a Broad But Selective Ligand Spectrum of a Mouse Olfactory Receptor: Mapping the Odorant-Binding Site

The olfactory receptor (OR) superfamily provides a basis for the remarkable ability to recognize and discriminate a large number of odorants. In mice, the superfamily includes ∼1000 members, and they recognize overlapping sets of odorants with distinct affinities and specificities. To address the molecular basis of odor discrimination by the mammalian OR superfamily, we performed functional analysis on a series of site-directed mutants and performed ligand docking simulation studies to define the odorant-binding site of a mouse OR. Our results indicate that several amino acids in the transmembrane domains formed a ligand-binding pocket. Although other G-protein-coupled receptors (GPCRs) recognize biogenic ligands mainly with ionic or hydrogen bonding interactions, ORs recognize odorants mostly via hydrophobic and van der Waals interactions. This accounts for the broad but selective binding by ORs as well as their relatively low ligand-binding affinities. Furthermore, we succeeded in rational receptor design, inserting point mutations in the odorant-binding site that resulted in predicted changes in ligand specificity and antagonist activity. This ability to rationally design the receptor validated the binding site structure that was deduced with our mutational and ligand docking studies. Such broad and specific sensitivity suggests an evolutionary process during which mutations in the active site led to an enormous number of ORs with a wide range of ligand specificity. The current study reveals the molecular environment of the odorant-binding site, and it further advances the understanding of GPCR pharmacology

Étude de la résistance en fatigue des matériaux bitumineux

Ce programme de recherche a pour but d’évaluer et caractériser le comportement en fatigue de deux enrobés bitumineux de même type GB20, mais confectionné avec deux bitumes différents. Un des enrobés étant formulés en laboratoire avec un bitume polymère PG64-28 et le deuxième prélevé en centrale d’enrobage et confectionné avec un bitume conventionnel PG58-28. Pour évaluer la résistance de ces enrobés, deux essais de caractérisation homogènes en traction/compression sont réalisés sur des éprouvettes cylindriques, un essai de module complexe dans le domaine de comportement viscoélastique linéaire (VEL), afin de déterminer sa performance et ses caractéristiques mécaniques et un essai de fatigue. Les essais de fatigue ont été effectués selon un mode de sollicitation à déformation imposée, et ce, pour différents niveaux d’amplitudes. Tous les essais ont été réalisés à une seule fréquence de sollicitation de 10Hz et à différentes températures (10, 20 et 30 °C) de manière à analyser son incidence sur la réponse en fatigue de l’enrobé. Après la présentation et la validation des résultats des essais réalisés, une analyse comparative selon la nature du bitume, l’influence de la température et la dispersion des durées de vie est exposée. Également, une méthode dite DGCB (Département de Génie Civil et Bâtiment) est appliquée pour l'analyse de la fatigue en calculant le taux d'endommagement par cycle suivi d’une étude menée sur les différents critères de rupture (Nf) visant à prédire la durée de vie en fatigue des enrobés bitumineux

Fruit scent and observer colour vision shape food-selection strategies in wild capuchin monkeys

The senses play critical roles in helping animals evaluate foods, including fruits that can change both in colour and scent during ripening to attract frugivores. Although numerous studies have assessed the impact of colour on fruit selection, comparatively little is known about fruit scent and how olfactory and visual data are integrated during foraging. We combine 25 months of behavioural data on 75 wild, white-faced capuchins (Cebus imitator) with measurements of fruit colours and scents from 18 dietary plant species. We show that frequency of fruit-directed olfactory behaviour is positively correlated with increases in the volume of fruit odours produced during ripening. Monkeys with red-green colour blindness sniffed fruits more often, indicating that increased reliance on olfaction is a behavioural strategy that mitigates decreased capacity to detect red-green colour contrast. These results demonstrate a complex interaction among fruit traits, sensory capacities and foraging strategies, which help explain variation in primate behaviour.https://www.nature.com/articles/s41467-019-10250-9Published versio

Amino acid coevolution reveals three-dimensional structure and functional domains of insect odorant receptors.

Insect odorant receptors (ORs) comprise an enormous protein family that translates environmental chemical signals into neuronal electrical activity. These heptahelical receptors are proposed to function as ligand-gated ion channels and/or to act metabotropically as G protein-coupled receptors (GPCRs). Resolving their signalling mechanism has been hampered by the lack of tertiary structural information and primary sequence similarity to other proteins. We use amino acid evolutionary covariation across these ORs to define restraints on structural proximity of residue pairs, which permit de novo generation of three-dimensional models. The validity of our analysis is supported by the location of functionally important residues in highly constrained regions of the protein. Importantly, insect OR models exhibit a distinct transmembrane domain packing arrangement to that of canonical GPCRs, establishing the structural unrelatedness of these receptor families. The evolutionary couplings and models predict odour binding and ion conduction domains, and provide a template for rationale structure-activity dissection

Extreme expansion of the olfactory receptor gene repertoire in African elephants and evolutionary dynamics of orthologous gene groups in 13 placental mammals

Olfactory receptors (ORs) detect odors in the environment, and OR genes constitute the largest multigene family in mammals. Numbers of OR genes vary greatly among species—reflecting the respective species\u27lifestyles—and this variation is caused by frequent gene gains and losses during evolution. However, whether the extent of gene gains/losses varies among individual gene lineages and what might generate such variation is unknown. To answer these questions, we used a newly developed phylogeny-based method to classify >10,000 intact OR genes from 13 placental mammal species into 781 orthologous gene groups (OGGs); we then compared the OGGs. Interestingly, African elephants had a surprisingly large repertoire (∼2000) of functional OR genes encoded in enlarged gene clusters. Additionally, OR gene lineages that experienced more gene duplication had weaker purifying selection, and Class II OR genes have evolved more dynamically than those in Class I. Some OGGs were highly expanded in a lineage-specific manner, while only three OGGs showed complete one-to-one orthology among the 13 species without any gene gains/losses. These three OGGs also exhibited highly conserved amino acid sequences; therefore, ORs in these OGGs may have physiologically important functions common to every placental mammal. This study provides a basis for inferring OR functions from evolutionary trajectory.UTokyo Research掲載「アフリカゾウはイヌの2倍もの嗅覚受容体遺伝子を持つ」 URI: http://www.u-tokyo.ac.jp/ja/utokyo-research/research-news/elephants-have-twice-as-many-olfactory-receptor-genes-as-dogs/UTokyo Research "Elephants have twice as many olfactory receptor genes as dogs" URI: http://www.u-tokyo.ac.jp/en/utokyo-research/research-news/elephants-have-twice-as-many-olfactory-receptor-genes-as-dogs

セグメントベツ ザイムホウコクキジュン ノ コクサイテキ コンバージェンス:ホウコクタイショウ セグメントシキベツキジュン ヲ メグッテ

我が国のセグメント情報は、1990年より連結財務諸表の注記情報として開示が制度化され、1998年度の全面適用により、我が国のセグメント会計基準の国際的調和を達成することができたといえる。しかし1997年に米国財務会計審議会(FASB)は、従来の基準であるSFAS第14号「企業のセグメント別財務報告」を改訂するSFAS第131号「企業のセグメント別情報ならびに関連情報の開示」を公表した。さらに、同年8月に国際会計基準委員会(IASC)は、IAS第14号「セグメント別財務情報の報告」を抜本的に見直したIAS第14号(改訂)「セグメント別報告」を公表し、さらにIASCを引き継いだIASBは、2006年にIFRS第8号「「オペレーティング・セグメント」を公表し、SFAS第131号とのコンバージェンスを達成した。そのため、我が国のセグメント会計基準と米国およびIASBとの基準間で新たな差異が発生したのである。我が国も本年9月に、企業会計基準公開草案第21号「セグメント情報等の開示に関する会計基準(案)」を公表し、国際的コンバージェンスを図っている。本稿では、セグメント会計基準の国際的コンバージェンスの方向と問題点についてセグメンテーションに焦点をあてて検討している。従来の産業セグメント・アプローチに代えて、マネジメント・アプローチを導入するということは、単にセグメンテーションの変更のみならず、セグメント情報の目的も、企業経営の多角化、グローバル化の状況を明らかにするという観点から、企業の過去の業績を理解し、将来キャシュ・フローの予測を適切に行うために、企業を構成する事業活動単位の状況を明らかにする分割情報を提供することへという開示目的の変化も意味している。国際的潮流であるマネジメント・アプローチを我が国に導入するにあたり、完全事業部制等の会社組織形態であればスムーズに対応することも実務上可能であるが、我が国に多いとされる職能別組織等他の形態を採用している場合、実務上の多くの困難を伴うことが予想される。会計基準設定において、国際的コンバージェンスへの配慮と我が国の独自性との間でいかにバランスを保つのかという課題が残されている