イ オ ン チ ャ ネ ル ス ー パ フ ァ ミ リ K チ ャ ネ ル の分子構造と機能 K チ ャ ネ ル 開 口 薬 血 管 に お + チ ャ ネ ル の分子構造と機能 K + チ ャ ネ ル 開 口 薬 血 管 に お け る 過 分 極 弛 緩 連 関 最近の知見 柳 澤 輝 行 東 北 大 学 医 部 分 子 薬 理

イ オ ン チ ャ ネ ル の 系 統 発 生 的 関 係 動 物 原 生 生 物 原 始 真 核 生 物 原 始 原 核 生 物 電 位 ( H i l l e , 1 9 9 2 よ り 改 変 ) C a 遊 A B C 二 回 膜 イ オ ン チ ャ ネ ル 内 蔵 型 電 位 依 存 性 離 輸 送 体 貫 通 型 n A C h G l u G l y G A B R y A C C F T R E N a C I R K c N K C a N a a b g d 動 物 N a ? 7 億 年 前 原 生 生 物 ? C a I P 3 2 x K 2 x 電 位 依 存 性 原 始 真 核 生 物 陽 イ オ ン 選 択 性 ? V D A C ? 1 4 億 年 前 ? 原 始 原 核 生 物 機 械 受 容 P o r i n A B C C o l i c i n s F o

The historical view. Potassium channels, key controllers of resting and action potentials (A) Clay Armstrong: Science 1998 280: 56-57.

イオンチャネルスーパーファミリーの構造と分類

Kチャネル（四量体）の構造

Science 1998 280: 69-77.

K+ channel’s pore (GYG) Sideview of K+ channel

M1/S5 M1/S5

K チャネルの系統発生的関係 +

a b Estrogen <BKCa> S t r u c t u r a l e l e m e n t s a n d f i o n s o f t h e K + c h a n n e l S t r u c a l d o m i n s C o r e - d m a i n P ( S S 1 - P o r e - f o r m i n g r e g i o n S S 2 , H 5 ) B l o c k e r s o f b i n d i n g s i t e s ( T E A , T B A , C T X , D T X ) O u t s i d e a + - s u b u n i t S 4 1 2 3 4 5 6 ( B a , 4 - A P ) 2 + V o l t a g e s e n s o r + S 6 / H M 1 M 2 I n s i d e S l o w i n a c t i v a t i o n g a t e N ' - d o m a i n + R e c o v e r y f r o m i n a c t i v a t i o n S 4 5 C O - 2 C ' - d o m a i n L i g a n d s b i n d i n g s i t e s S t r u c a l e m n s H N + 3 R e c e p t o r f o r f a s t i n a c t i v a t i o n b a l l F u n c t i o n s F a s t i n a c t i v a t i o n b a l l N A D P T e t r a m e r a s s e m b l y Estrogen <BKCa> P O 2 H e m e R e d o x s t a t e b - s u b u n i t A T P / A D P I n w a r d e c t i f s : N ' , M 1 H 5 2 & C

K+ channel was thought to be "long" pores, wide at the ends with a narrow selectivity filter (B) that was a good fit for hydrated ions (C and E) and dehydrated K+ (D), but a poor fit for Na+ (F).

Topview of K+ channel Science 1998 280: 69-77.

Science 1998 280: 69-77.

種々の遮断薬blockersにより閉じる。 どのような機序を君は思いつくか？ + 　 K チャネルは不活性化と 種々の遮断薬blockersにより閉じる。 どのような機序を君は思いつくか？ K+　current Control Inactivation ・II・ Blocker B Blocker A 0.1 sec

a b Estrogen <BKCa> S t r u c t u r a l e l e m e n t s a n d f i o n s o f t h e K + c h a n n e l S t r u c a l d o m i n s C o r e - d m a i n P ( S S 1 - P o r e - f o r m i n g r e g i o n S S 2 , H 5 ) B l o c k e r s o f b i n d i n g s i t e s ( T E A , T B A , C T X , D T X ) O u t s i d e a + - s u b u n i t S 4 1 2 3 4 5 6 ( B a , 4 - A P , Quinidine, Verapamil) 2 + V o l t a g e s e n s o r + S 6 / H M 1 M 2 I n s i d e S l o w i n a c t i v a t i o n g a t e N ' - d o m a i n + R e c o v e r y f r o m i n a c t i v a t i o n S 4 5 C O - 2 C ' - d o m a i n L i g a n d s b i n d i n g s i t e s S t r u c a l e m n s H N + 3 R e c e p t o r f o r f a s t i n a c t i v a t i o n b a l l F u n c t i o n s F a s t i n a c t i v a t i o n b a l l N A D P T e t r a m e r a s s e m b l y Estrogen <BKCa> P O 2 H e m e R e d o x s t a t e b - s u b u n i t A T P / A D P I n w a r d e c t i f s : N ' , M 1 H 5 2 & C

Agatoxin S t r u c e o f c h a r y b d o t x i n h e l i x a b s h e t + + 1 7 h e l i x a b s h e t 2 3 5 + 3 3 N H 2 + + + 1 3 C O O H + - 2 8 + ( b ) 7

ペプチド性Kチャネル遮断薬は細胞外からチャネルのポアをちょうど 栓をするように塞いで遮断する。 チャネルポア Science 1998 280: 106-109.

Fig. 3: Structure of Ion Channels Fig. 3: Structure of Ion Channels. Panel A shows a subunit containing six transmembrane-spanning motifs, S1 through S6, that forms the core structure of sodium, calcium, and potassium channels. Panel B shows four such subunits assembled to form a potassium channel. The pore region appears to have wide intracellular and extracellular vestibules (approximately 2.8 to 3.4 nm wide and 0.4 to 0.8 nm deep) that lead to a constricted pore 0.9 to 1.4 nm in diameter at its entrance, tapering to a diameter of 0.4 to 0.5 nm at a depth of 0.5 to 0.7 nm from the vestibule.

Postulated Location of the Quinidine-Binding Site within the Transmembrane Segment Responsible for the Blockade of a Delayed Rectifier Potassium Channel (hKv1.5). A side view of the S6 helix shows that residues T505 and V512, which have been implicated in quinidine binding, are aligned on the same side of the helix. New England Journal of Medicine -- January 1, 1998 -- Vol. 338, No. 1

N Verapamil N-methyl-verapamil Structure of the phenylalkylamines verapamil and N-methyl-verapamil. (A) Chemical structure of verapamil (RII=H) and the permanently charged-quarternary N-methyl-verapamil (RII=CH3). (B) Three-dimensional structure of verapamil (spacefill).

Yellow: phenylalkylamine binding site Gray: homologous amino acids identical to S6 residues

Side view of the K channel with verapamil GYGD-motif (sticks) PAA binding sites Verapamil T396 & T397 (sticks) Shown are three subunits of the KcsA channel as ribbons.

View from above into the channel pore. The protonated nitrogen of verapamil is visible in the center of the pore facing towards the negative environment of the GYGD-motif. The phenyl rings are located in a hydrophobic environment in closest proximity to the residues homologous to M395 and G421.

+ 1) K チャネルは種々の細胞機能に 影響する。 + 2) K チャネル開口薬の作用は過分 極がその機序にある。 + 3) K チャネル開口薬は間接的な Ca拮抗薬ではない。 4) 弛緩機序を総合して過分極弛緩連 関と呼ぶ。

+ 1) K チャネルは種々の細胞機能に 影響する。 どのような機能を君は思いつくか？ 例．神経の膜電位の再分極 担う機能が多彩であるとすれば、．．

心筋の膜電位・膜電流・イオンチャネルの概観 　　　　　心筋の膜電位・膜電流・イオンチャネルの概観　　　　　 オーバーシュート イオン濃度、平衡電位、電流の方向と膜電位効果 プラトー 膜電流 膜電位 細胞外　 細胞内　平衡電位　方向　効果 ノッチ Na + 145 mM 10 mM 70 mV 内向き　脱分極 0 mV ウィンドウ電流 Ca 2+ 2 mM 100 nM 129 mV 内向き　脱分極 内向き電流 再分極 2 nA K + 4 mM 150 mM -94 mV 外向き　再分極 0.5 nA a b c 過分極 静止膜電位 -85 mV 外向き電流 20 nA 100 200 300 400 時間　(msec) 遮断薬 Na + 電位依存性 チャネル 局所麻酔薬、テトロドトキシン（TTX） Ca 2+ 電位依存性 チャネル Ca拮抗薬 K + 電位依存性 チャネル III群抗不整脈薬、テトラエチルアンモニウム（TEA） K + 一過性外向き チャネル (b) K + 遅延整流 チャネル (c) K + 内向き整流 チャネル (a) ； K ； K アトロピン；　グリベンクラミド ACh ATP

The prolonged QT interval as measured on the ECG results from an increased duration of the cardiac AP (Panel B). The ventricular AP is maintained at a resting membrane potential (approximately -85 mV) by inwardly rectifying K+ currents (IK1, phase 4). Once an excitatory stimulus depolarizes the cell beyond a threshold voltage (for example, -60 mV), Na+ currents are activated that quickly depolarize the cell (INa, phase 0). These Na+ channels are rapidly inactivated, allowing transient K+ currents to return the AP to the plateau voltage (phase 1). The plateau lasts about 300 msec and provides time for the heart to contract. The plateau is maintained by the competition between outward-moving K+ currents and inward-moving Ca2+ currents (phase 2). Progressive inactivation of Ca2+ currents and increasing activation of K+ currents repolarize the cell to the resting membrane potential (phase 3).

Copyright © 1997 by the Massachusetts Medical Society. All rights reserved.

ヘテロマルチマーKATPチャネルのサブユニット構造 タ イ プ S U R S U R K i r S U R 1 + K i r 6 . 2 b 細 胞 K i r K i r * K i r S U R 2 A + K i r 6 . 心 筋 S U R S U R S U R 2 B + K i r 6 . 平 滑 筋 S U R 2 B + K i r 6 . 1 平 滑 筋 ( K N D P チ ャ ネ ル ) H N 2 * 細 胞 外 ジ ア ゾ キ サ イ ド 結 合 部 位 M 1 M 2 細 胞 内 C O O H A T P A B 結 合 部 位 W a l k e r A B H N 2 N B F - 1 N B F - 2 M g - A D P C O O H 結 合 部 位 A T P 感 受 性 決 定 不 活 性 化 部 位

O O O

Ca signaling in vascular smooth muscle IP receptor 3 Ryanodine receptor Ca channels 2+ Contraction Ca 2+ Ca 2+ wave Agonists Ca 2+ G q PKC Ca 2+ DG Ca 2+ G Ca 2+ R PLC Ca 2+ Ca 2+ Ca 2+ Ca 2+ Ca 2+ Ca 2+ Ca 2+ IP 3 IP 3 IP 3 IP 3 IP 3 Ca 2+ Ca 2+ Ca 2+ SR Ca pump Ca 2+ Ca 2+ Ca 2+ 3Na + 2K Ca 2+ K Ca K + Ca 2+ Na-Ca exchanger 3Na + Ca sparks 2+ Na pump STOC Sarcolemma Relaxation Ca pump STOCs: Spontaneous Transient Outward (hyperpolarizing ) Currents : Ca -activated K channels 2+ + K Ca

+ 1) K チャネルは種々の細胞機能に 影響する。 + 2) K チャネル開口薬の作用は過分 極がその機序にある。 + 3) K チャネル開口薬は間接的な Ca拮抗薬ではない。 4) 弛緩機序を総合して過分極弛緩連 関と呼ぶ。

Pyrimidine: minoxidil, LP 805 O N H 2 発毛剤

膜電位 （mV） K チャネル Ca チャネル 血管平滑筋 の緊張 血管径 収縮刺激・ 脱分極 静止時 過分極 高血圧 －40 －55 －75 K ＋ 平衡電位 －90 細胞外 K ＋ 細胞膜 チャネル 閉 K ＋ ＋ ＋ 開 K K K ＋ K ＋ K ＋ Ca 2＋ Ca 2＋ Ca 2＋ Ca 2＋ Ca 2＋ Ca 2＋ Ca 2＋ 細胞膜 チャネル 開 細胞内 閉 血管平滑筋 過分極弛緩連関 の緊張 血管径 K ＋ チャネルオープナー

脳血流の自己調節 autoregulation 300 血流量 200 (ml/min) 100 100 200 100 200 潅流圧（動静脈圧差　(mmHg)

静止膜電位 (mV) 単一細胞で開いているＫチャネルの数 E = BK , IK ：Ｃａ活性化Ｋチャネル Ca Ca K ATP -90 -80 -70 -60 -50 静止膜電位　(mV) 100 80 60 40 20 150 pS 10 pS 20 pS 40 pS 60 pS 100 pS 単一コンダクタンス 単一細胞で開いているＫチャネルの数 E K = BK , IK ：Ｃａ活性化Ｋチャネル Ca Ca 約100 pS, 約60 pS K ATP ：ATP感受性Ｋチャネル 約100 pS, 約10 pS ：電位依存性Ｋチャネル Kv 約10 pS

+ 1) K チャネルは種々の細胞機能に 影響する。 + 2) K チャネル開口薬の作用は過分 極がその機序にある。 + 3) K チャネル開口薬は間接的な Ca拮抗薬ではない。 4) 弛緩機序を総合して過分極弛緩連 関と呼ぶ。

K c h a n e l o p r i b t s C f m S R y x A g u + 2 C a U 4 6 6 1 9 C C a U 4 6 6 1 9 C a f f e i n e . 6 F u r a - 2 r a t i o . 5 C o n t r o l 3 C r o m a k l i 1 M - 5 F o r c e ( m N ) 1 m i n

2 4 6 8 1 F o r c e ( % ) [ C a ] + i - D s n l V p m M Ca　sensitivity and the concentration of KCl (Vm) 2 4 6 8 1 F o r c e ( % ) [ C a ] + i - 5 7 . 9 K 3 D s n l V p m M

K channel openers [Ca ] Force Relaxation mechanisms of K+ channel openers K channel openers + Agonists 2+ Ca channel Receptor Hyperpolarization Hyperpolarization G q K ATP PLC DG GTP GDP K + K + IP 3 <PKC ／rho> Hyperpolarization Ca 2+ SR 2+ i [Ca ] 2+ Ca sensitivity Force

Mechanisms of Disease: The Protective Effects of Estrogen on the Cardiovascular System New England Journal of Medicine -- June 10, 1999 -- Vol. 340, No. 23 p.1801-1811 Estrogen has both rapid and longer-term effects on the blood-vessel wall. The mechanisms that mediate the rapid effects of estrogen are not fully understood. Current data suggest that estrogen influences the bioavailability of endothelial-derived nitric oxide and, through nitric oxide-mediated increases in cGMP, causes the relaxation of vascular smooth-muscle cells. The longer-term effects of estrogen, about which more is known, are due at least in part to changes in vascular-cell gene and protein expression that are mediated by estrogen receptor (alpha), (beta), or both. Estrogen-regulate proteins influence vascular function in an autocrine or paracrine fashion. However, additional vascular target genes regulated by estrogen receptors need to be identified.

Fig. 4: Mechanism of rapid, nongenomic activation of nitric oxide synthase by estrogen in endothelial Cells and vascular smooth-muscle cells.

Direct Effects on Vascular Cells & Tissues- Rapid, Nongenomic Effects Estrogens can cause short-term vasodilatation by both endothelium-dependent and endothelium-independent pathways. These rapid effects do not appear to involve changes in gene expression. Two mechanisms for the rapid vasodilatory effects of estrogens have been explored in some depth: effects on (1) ion-channel function and (2) effects on nitric oxide. (1) ion-channel function calcium-activated potassium channels (At physiologic concentrations) L-type calcium channels (high concentrations of estrogen) (2) effects on nitric oxide Estrogen receptor (alpha) can directly activate endothelial nitric oxide synthase, perhaps through a tyrosine kinase pathway or the mitogen-activated protein kinase signaling pathway; may involve proteins that interact with the estrogen receptor, such as heat-shock protein 90, which also binds to and activates endothelial nitric oxide synthase The short-term coronary vasodilatory effects of estrogen in humans are largely mediated by the increased production of nitric oxide.

Maximizing the benefits of K+ channels Maximizing the benefits of K+ channels. The Maxi K+ channel of vascular smooth muscle cells is composed of both a and b subunits (top), whereas that of skeletal muscle cells is composed of a subunits alone (bottom). 17b-Estradiol binds to and increases the activity of Maxi K+ channels with b subunits. The resulting efflux of K+ from the vascular smooth muscle cells results in closure of Ca2+ channels and relaxation of the muscle in the blood vessel wall. Science Volume 285, Number 5435 Issue of 17 Sep 1999, p 1859

Acute Activation of Maxi-K Channels (hSlo) by Estradiol Binding to the b Subunit Valverde MA et al. Science Volume 285, Number 5435, pp. 1929 - 1931 Maxi-K channels consist of a pore-forming subunit and a regulatory b subunit, which confers the channel with a higher Ca2+ sensitivity. Estradiol bound to the subunit and activated the Maxi-K channel (hSlo) only when both a and b subunits were present. This activation was independent of the generation of intracellular signals and could be triggered by estradiol conjugated to a membrane-impenetrable carrier protein. This study documents the direct interaction of a hormone with a voltage-gated channel subunit and provides the molecular mechanism for the modulation of vascular smooth muscle Maxi-K channels by estrogens.

Figure 4. Effect of 17b-estradiol on Maxi-K channels reconstituted in lipid bilayers. Channels obtained from skeletal muscle (A), smooth muscle (B), or Xenopus oocytes expressing and subunits (C) were recorded at 30 mV, 60 mV, and 40 mV, respectively, before and after the addition of 4.2 mM 17b-estradiol to the external side of the membrane.