野辺山高原におけるヤエガワカンバ林の植物社会学的研究

[論文

非不活化カリウム電流の発現を増強する膜蛋白KCR1は,ラットEAG電位依存性カリウムチャンネルと結合する

取得学位 : 博士（医学）, 学位授与番号 : 医博乙第1467号, 学位授与年月日：平成11年1月20日,学位授与年：199

Caオシレーション時におこるCa流入チャンネルの同定

金沢大学医学部研究課題/領域番号:04857010, 研究期間(年度):1992出典：研究課題「Caオシレーション時におこるCa流入チャンネルの同定」課題番号04857010（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-04857010/）を加工して作

電位依存性カリウムMチャンネルの受容体による制御機構の解明

金沢大学医薬保健研究域医学系受容体刺激によるMチャンネル抑制は、神経興奮性を制御する重要な機構の一つであるが、このシグナル伝達系については十年来混乱している。我々は近年Mチャンネル本体と同定されたKCNQ2, 3チャンネルを用いてムスカリン作動性アセチルコリン受容体(M_1)刺激からのシグナル伝達経路を解析した。今回特にAKAP150がプロテインキナーゼC(PKC)、カルシネウリン、カルモジュリンと結合することに注目し、KCNQ2/3チャンネル修飾におけるAKAP150の関与を、部分欠損変異体を用いて解析した。PKC結合部位欠損変異体において、受容体によるチャンネル修飾が消失しこれらの複合体がなんらかの役割をはたしていることが明らかになった。更にこの複合体を解析するため、filter overlay法によりチャンネルとAKAP150の相互作用を調べたところ両者が直接結合することが分かった。従来、PKCの関与は、PKC阻害薬が受容体修飾を解除できないことから否定的と考えられていた。数種類のPKC阻害剤を投与したところ、PKCの活性中心に結合するbisindolylmaleimide、chelerythrineは効果がなく、ジアシルグリセロール結合領域に作用するcalphostin、safingolだけがM電流抑制を解除した。AKAP150がPKCの活性中心に結合することが知られているので、複合体形成により、ある種のPKC阻害剤の標的部位が保護されていると考えられた。このことを検証するため、^Pを用いた代謝ラベル法によりチャンネルのリン酸化を測定したところ、KCNQ2チャンネルのリン酸化は、電気生理と同様の結果が得られ、複合体形成によりPKC阻害剤の感受性が変化することが分かった。以上、今回の研究からMチャンネルはAKAP150と複合体を形成しその構成要素のPKCにより制御されていることが明らかになった。研究課題/領域番号:12770017, 研究期間(年度):2000-2001出典：「電位依存性カリウムMチャンネルの受容体による制御機構の解明」研究成果報告書　課題番号 12770017（KAKEN：科学研究費助成事業データベース（国立情報学研究所））（https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-12770017/)を加工して作

小脳由来の不活性化しないカリウムチャネルの分子機構

金沢大学医薬保健研究域医学系小脳より抽出したmRNAを注入して不活化しないK電流(IK(ni))をアフリカツメガエル卵母細胞に再構成すると、1996年にWatkins,Marthieらによって記載されたIK(SO)と良く似た電流が観察された。即ち、-60mV以上の深い膜電位から活性化が始まり、20分以上にわたって不活化せず、細胞内Ca上昇を伴うホルモン刺激で一過性に抑制された。本研究では、IK(ni)の分子情報を得るため、遺伝子クローニングを実施し小脳cDNAライブラリーより1つの遺伝子(KCR1)を得た。この遺伝子がコードしていたタンパクは、12回の膜貫通領域をもち、酵母、線虫のゲノムプロジェクトでみつかったものと20-35%の相同性をもっていたが、ほ乳類ではまだ知られていない新規のタンパクであった。この遺伝子cRNAをアフリカツメガエル卵母細胞に注入したが、チャンネル活性は認められなかった。そこで、このタンパクが、Kチャンネルの制御タンパクであろうと仮定し、小脳で発現している既知の電位依存性Kチャンネルと卵母細胞に共発現し、電流性質変化の有無を調べた。この実験から、ラットeagKチャンネルの性質を変えることがわかった。ラットeagKチャンネルは、細胞外Mg存在下で、ゆっくり(100msec)と活性化するが、KCR1と共発現すると、この活性化が速くなることがわかった。生化学的に2つのタンパクが、結合しているかを、免疫沈降とFar-westemプロッティングで確かめたところ、これらは複合体を形成していることが確認でき、このことは電気生理のデータを支持した。抗体を作成し、脳内分布を調べたところKCR1は、小脳顆粒細胞層、分子層、海馬CA3領域に多く局在していた。eagは、ほぼ同一部位に局在し、脳内でこれらが複合体を形成し機能していることがわかった。研究課題/領域番号:09780721, 研究期間(年度):1997 – 1998出典：「小脳由来の不活性化しないカリウムチャネルの分子機構」研究成果報告書　課題番号09780721（KAKEN：科学研究費助成事業データベース（国立情報学研究所））（https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-09780721/)を加工して作

Case report: Neonatal case of intrauterine gastrointestinal bleeding with suspected cow's milk allergy or neonatal transient eosinophilic colitis

The patient was a female newborn. Ultrasonography performed at 35 weeks and 3 days of gestation revealed honeycomb-like dilatation and peri-intestinal strong echo patterns in the gastrointestinal tract. Nonreassuring fetal status was also diagnosed, leading to an emergency Cesarean section. The baby's birth weight was 2,127 g, whereas the Apgar 1 min and 5 min scores were 8 and 9, respectively. The amniotic fluid showed fecal and hematogenous turbidity. After delivery, there was hematogenous intragastric residue and defecation. Thereafter, the bloody intragastric residue and fecal discharge improved. Aggregations of eosinophils in the stool were observed, and gastrointestinal allergy was suspected. Enteral feeding with the hydrolyzed protein formula was initiated and symptoms did not recur. The allergen-specific lymphocyte stimulation test was positive for lactoferrin, and the patient was suspected with neonatal cow's milk allergy or neonatal transient eosinophilic colitis. After her condition stabilized, an oral challenge test was performed using breast milk without dairy products, and the test was negative. Gastrointestinal allergy is rare even in utero, and when gastrointestinal bleeding is suspected in utero, hemorrhagic or surgical gastrointestinal diseases should be ruled out first; however, the possibility of gastrointestinal allergy should also be kept in mind

Insulin receptor substrate-2 maintains predominance of anabolic function over catabolic function of osteoblasts

Insulin receptor substrates (IRS-1 and IRS-2) are essential for intracellular signaling by insulin and insulin-like growth factor-I (IGF-I), anabolic regulators of bone metabolism. Although mice lacking the IRS-2 gene (IRS-2−/− mice) developed normally, they exhibited osteopenia with decreased bone formation and increased bone resorption. Cultured IRS-2−/− osteoblasts showed reduced differentiation and matrix synthesis compared with wild-type osteoblasts. However, they showed increased receptor activator of nuclear factor κB ligand (RANKL) expression and osteoclastogenesis in the coculture with bone marrow cells, which were restored by reintroduction of IRS-2 using an adenovirus vector. Although IRS-2 was expressed and phosphorylated by insulin and IGF-I in both osteoblasts and osteoclastic cells, cultures in the absence of osteoblasts revealed that intrinsic IRS-2 signaling in osteoclastic cells was not important for their differentiation, function, or survival. It is concluded that IRS-2 deficiency in osteoblasts causes osteopenia through impaired anabolic function and enhanced supporting ability of osteoclastogenesis. We propose that IRS-2 is needed to maintain the predominance of bone formation over bone resorption, whereas IRS-1 maintains bone turnover, as we previously reported; the integration of these two signalings causes a potent bone anabolic action by insulin and IGF-I

Respiratory mechanics and cerebral blood flow during heat‐induced hyperventilation and its voluntary suppression in passively heated humans

We investigated whether heat‐induced hyperventilation can be voluntarily prevented, and, if so, how this modulates respiratory mechanics and cerebral blood flow in resting heated humans. In two separate trials, 10 healthy men were passively heated using lower body hot‐water immersion and a water‐perfused garment covering their upper body (both 41°C) until esophageal temperature (Tes) reached 39°C or volitional termination. In each trial, participants breathed normally (normal‐breathing) or voluntarily controlled minute ventilation (VE) at a level equivalent to that observed after 5 min of heating (controlled‐breathing). Respiratory gases, middle cerebral artery blood velocity (MCAV), work of breathing, and end‐expiratory and inspiratory lung volumes were measured. During normal‐breathing, VE increased as Tes rose above 38.0 ± 0.3°C, whereas controlled‐breathing diminished the increase in VE (VE at Tes = 38.6°C: 25.6 ± 5.9 and 11.9 ± 1.3 L min−1 during normal‐ and controlled‐breathing, respectively, P < 0.001). During normal‐breathing, end‐tidal CO2 pressure and MCAV decreased with rising Tes, but controlled‐breathing diminished these reductions (at Tes = 38.6°C, 24.7 ± 5.0 vs. 39.5 ± 2.8 mmHg; 44.9 ± 5.9 vs. 60.2 ± 6.3 cm sec−1, both P < 0.001). The work of breathing correlated positively with changes in VE (P < 0.001) and was lower during controlled‐ than normal‐breathing (16.1 ± 12.6 and 59.4 ± 49.5 J min−1, respectively, at heating termination, P = 0.013). End‐expiratory and inspiratory lung volumes did not differ between trials (P = 0.25 and 0.71, respectively). These results suggest that during passive heating at rest, heat‐induced hyperventilation increases the work of breathing without affecting end‐expiratory lung volume, and that voluntary control of breathing can nearly abolish this hyperventilation, thereby diminishing hypocapnia, cerebral hypoperfusion, and increased work of breathing

M-Current Suppression, Seizures and Lipid Metabolism: A Potential Link Between Neuronal Kv7 Channel Regulation and Dietary Therapies for Epilepsy.

Neuronal Kv7 channel generates a low voltage-activated potassium current known as the M-current. The M-current can be suppressed by various neurotransmitters that activate Gq-coupled receptors. Because the M-current stabilizes membrane potential at the resting membrane potential, its suppression transiently increase neuronal excitability. However, its physiological and pathological roles in vivo is not well understood to date. This review summarizes the molecular mechanism underlying M-current suppression, and why it remained elusive for many years. I also summarize how regulation of neuronal Kv7 channel contributes to anti-seizure action of valproic acid through inhibition of palmitoylation of a Kv7 channel binding protein, and discuss about a potential link with anti-seizure mechanisms of medium chain triglyceride ketogenic diet

A change in configuration of the calmodulin-KCNQ channel complex underlies Ca2+-dependent modulation of KCNQ channel activity.

All subtypes of KCNQ channel subunits (KCNQ1-5) require calmodulin as a co-factor for functional channels. It has been demonstrated that calmodulin plays a critical role in KCNQ channel trafficking as well as calcium-mediated current modulation. However, how calcium-bound calmodulin suppresses the M-current is not well understood. In this study, we investigated the molecular mechanism of KCNQ2 current suppression mediated by calcium-bound calmodulin. We show that calcium induced slow calmodulin dissociation from the KCNQ2 channel subunit. In contrast, in homomeric KCNQ3 channels, calcium facilitated calmodulin binding. We demonstrate that this difference in calmodulin binding was due to the unique cysteine residue in the KCNQ2 subunit at aa 527 in Helix B, which corresponds to an arginine residue in other KCNQ subunits including KCNQ3. In addition, a KCNQ2 channel associated protein AKAP79/150 (79 for human, 150 for rodent orthologs) also preferentially bound calcium-bound calmodulin. Therefore, the KCNQ2 channel complex was able to retain calcium-bound calmodulin either through the AKPA79/150 or KCNQ3 subunit. Functionally, increasing intracellular calcium by ionomycin suppressed currents generated by KCNQ2, KCNQ2(C527R) or heteromeric KCNQ2/KCNQ3 channels to an equivalent extent. This suggests that a change in the binding configuration, rather than dissociation of calmodulin, is responsible for KCNQ current suppression. Furthermore, we demonstrate that KCNQ current suppression was accompanied by reduced KCNQ affinity toward phosphatidylinositol 4,5-bisphosphate (PIP2) when assessed by a voltage-sensitive phosphatase, Ci-VSP. These results suggest that a rise in intracellular calcium induces a change in the configuration of CaM-KCNQ binding, which leads to the reduction of KCNQ affinity for PIP2 and subsequent current suppression