Transcript Document

イオンチャネルスーパーファミリー
+
K
チャネルの分子構造と機能
K+ チャネル開口薬
血管における過分極弛緩連関
最近の知見
柳澤 輝行
東北大学医学部分子薬理
イオンチャネルの系統発生的関係
二回膜
Ca
ABC
イオンチャネル内蔵型 離 遊 輸送体 貫通型
nACh Glu Gly GABAA Ry
AC CFTR ENaC IR K cN
(Hille,1992より改変)
電位依存性
K
a b g d
Ca
Na
動物
Na
?
7億年前
原生生物
?
Ca
IP3
K
2x
2x
電位依存性
原始真核生物
陽イオン選択性
?
?
14億年前
VDAC
?
ABC
機械受容
Porin
Colicins
Fo
原始原核生物
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
ス
・
パ
・
フ
ァ
ミ
リ
・
N
TRP
TRPL
C
C a 2 + a nd N a + C han nels
K + C ha nn els
C
N
6T M
N
サ
ブ
フ
ァ
ミ
リ
・
N
K + C han nels
構
造
型
フ
ァ
ミ
リ
・
他のイ オン チャ ネ ル
機械受容
ア ミ ロ ラ イ ド 感受性
P 2X 受 容 体
C
6T M + 2TM
N
C
V ol tag e-G ated
－ －
－
+ －
+
+
+
+
－ －
－
+
T1
N
C
N
S lo (K C a)
E ag
+
+
+
+
－
C
－
－ －
－
+ －
+
+ +
+
+
+
N
－
C
N
C a 2+
C a2+ ?
S hak er S hab S haw S hal
( K v 1) ( K v 2) (K v 3 ) ( K v 4)
nK Q T2
eag
S lo
erg el k
(herg )
メ ン バ ー K v 1 .1 K v 1.2 K v 1 .3 K v 1.4 K v 1.5 K v 1.6 K v 1.7 K v 1.8
nS lo2
+
+
+
N
cA M P
（ 嗅覚）
cG M P
（ 視覚）
C
S K Ca
CN G
－ －
－
－ －
C
K V L QT
N
N
C
TW I K T R E K TB A K
酵 母 C
K QT
2T M
4TM
－ －
+ +
+
－ －
－ － －
cN TP
C
N
C
K i r1 K i r2 K i r3 K ir4K ir5 K i r6 nIR K
K ir3.1 K ir3 .2 K ir3.3 K ir3.4
Kチャネル（四量体）の構造
内向き整流型Ｋチャネル
電位依存性Ｋチャネル
X2 & X2 Ｃａ、Ｎａチャネル
P
P
＋
＋
M1
S1 S2 S3 S4
＋
M2
S5
＋
S6
COOH
H 2N
H 2N
M1
M2
P
S3
S4
COOH
S2
S1
S5
S6
P
Science 1998 280: 69-77.
Sideview of K+ channel
K+ channel’s pore (GYG)
M1/S5
M1/S5
+
K チャネルの系統発生的関係
Structural elements and functions of the K+ channel
Structural
domains
P (SS1SS2, H5)
Core-domain
Outside
a -subunit
+
S4
1
2
3
Voltage sensor
Inside
Pore-forming region
Blockers of binding sites
(TEA, TBA, CTX, DTX)
4
+
5
M1
6 (Ba2+ , 4-AP)
M2
N'-domain
S45
S6/H6
Slow inactivation gate
Recovery from inactivation
CO2 -
C'-domain
Ligands binding sites
Structural
elements
Functions
+H N
3
Receptor for fast
inactivation b a l l
Fast inactivation ball
Tetramer assembly
NADP
PO2
Redox state
ATP/ADP
Heme
Estrogen
<BKCa>
b -subunit
Inward rectifiers: N', M1, H5, M2, & 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.
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
Structural elements and functions of the K+ channel
Structural
domains
P (SS1SS2, H5)
Core-domain
Outside
a -subunit
+
S4
1
2
3
Voltage sensor
Inside
Pore-forming region
Blockers of binding sites
(TEA, TBA, CTX, DTX)
4
+
5
M1
6 (Ba2+ , 4-AP, Quinidine, Verapamil)
M2
N'-domain
S45
S6/H6
Slow inactivation gate
Recovery from inactivation
CO2 -
C'-domain
Ligands binding sites
Structural
elements
Functions
+H N
3
Receptor for fast
inactivation b a l l
Fast inactivation ball
Tetramer assembly
NADP
PO2
Redox state
ATP/ADP
Heme
Estrogen
<BKCa>
b -subunit
Inward rectifiers: N', M1, H5, M2, & C'
Structure of
charybdotoxin
+
17
a helix
35
+
2 b sheets
+
33
NH2
+
+
+
13
COOH
+
-
(b)
28
+
7
Agatoxin
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
Kチャネル遮断薬
Science 1998 280: 106-109.
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.
Open channel block model
200 s -1
C
50 s -1
k (M -1 s -1 )
O
B
L (s -1 )
1/  D (s -1 ) = k [Drug] + L
K d = L/k, 120
M
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-methylverapamil (RII=CH3).
(B) Three-dimensional structure of verapamil
(spacefill).
Yellow: phenylalkylamine binding site
Gray: homologous amino acids identical to S6 resid
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.
The protonated
View from above into the channel pore.
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
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
イオン濃度、平衡電位、電流の方向と膜電位効果
オーバーシュート
プラトー
膜電位
膜電流
ノッチ
+
Na
0 mV
ウィンドウ電流
細胞外
細胞内
145 mM 10 mM
2+
Ca
内向き電流
c
b
a
2 mM 100 nM 129 mV 内向き
脱分極
4 mM 150 mM -94 mV 外向き
再分極
過分極
再分極
2 nA
+
K
平衡電位 方向 効果
70 mV 内向き 脱分極
0.5 nA
静止膜電位
外向き電流
-85 mV
20 nA
0
100
200
時間 (msec)
電位依存性 Na+ チャネル
2+
電位依存性 Ca チャネル
+
電位依存性 K チャネル
400
300
遮断薬
局所麻酔薬、テトロドトキシン（TTX）
Ca拮抗薬
III群抗不整脈薬、テトラエチルアンモニウム（TEA）
+
一過性外向き K チャネル(b)
+
遅延整流 K チャネル (c)
+
； K ATP
内向き整流 K チャネル(a)； K ACh
アトロピン； グリベンクラミド
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 inwardmoving 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
SUR
Kir
Kir
SUR
*Kir
サブユニット タイプ
SUR1 + Kir6.2 b 細胞
SUR2A+ Kir6.2 心筋
SUR
Kir
SUR
SUR2B+ Kir6.2 平滑筋
SUR2B+ Kir6.1 平滑筋
(KNDP チャネル)
H2N
*
細胞外
ジアゾキサイ
ド結合部位
細胞内
COOH
Walker A
B
NBF-1
ATP 感受性決定
A
B
H2N
NBF-2 Mg-ADP
結合部位
M1
M2
ATP
結合部位
COOH
不活性化部位
O
O
O
Ca signaling in vascular smooth muscle
Ca2+ channels
Ryanodine receptor
IP3 receptor
Contraction
Ca
Agonists
Gq
G R
PLC DG
2+
Ca 2+ wave
2+
Ca
2+
Ca
PKC
2+
2+
2+
Ca
Ca
Ca
IP3
IP3
2+
Ca Ca
2+
IP3
2+
2+
Ca Ca
IP3
IP3
2+
2+
Ca
2+
Ca Ca
2+
Ca
2+
Ca
2+
SR Ca pump
2+
Ca Ca
2+
+
Ca
3Na
2+
Ca
+
3Na
Na-Ca exchanger
KCa K
+
2+
Ca
+
2K
Na pump
Relaxation
Sarcolemma
Ca pump
STOC
Ca 2+ sparks
STOCs: Spontaneous Transient
Outward (hyperpolarizing ) Currents
KCa : Ca 2+ -activated K+ channels
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 + チャネル開口薬の化学構造式
Pyridine:
ニコランジル
KRN2391
,
C ON H C H 2 C H 2 ON O 2
NO, cGMP ↑
Pyrimidine:
N
H2 N
N
N
Benzopyran:
レブクロマカリム
発毛剤
O
N
NC
*
*
OH
CH3
O
CH3
ジアゾキサイド
minoxidil, LP 805
O
N
"N-K hybrid"
Benzothiadiazine:
NH2
N
CH
NH
Cl
S
O2
"Nonspecific KCO"
3
脱分極
膜電位
－40
（mV）
収縮刺激・
静止時
高血圧
－55
K ＋平衡電位
K＋
チャネル
Ca 2＋
チャネル
過分極
－75
－90
細胞外
細胞膜
閉
K＋
K＋
Ca 2＋Ca 2＋Ca 2＋ Ca 2＋
Ca 2＋
開K ＋
K＋
K＋
K＋
Ca 2＋
細胞膜
開
細胞内 閉
血管平滑筋
の緊張
過分極弛緩連関
血管径
K ＋チャネルオープナー
a 全身循環の細動脈
b 肺細動脈
低酸素、虚血、アデノシン
Ｋ + チャネル開口薬
肺胞内低酸素
血管内圧上昇
K ATP
（伸展受容チャネル開口）
脱分極
Kv and/or K Ca,ATP
の 抑制
抑制
脱分極
Kv
（電位依存性）
BK
Ca ,
IK Ca
（電位依存性）
電位依存性
Ｃａ2+ チャネルを
通してのＣａ流入
BK
Ca ,
IK Ca
（電位依存性）
電位依存性
Ｃａ2+ チャネルを
通してのＣａ流入
細胞内Ｃａ濃度上昇
細胞内Ｃａ濃度上昇
筋原性張力（収縮）
低酸素性肺血管収縮
血流自己調節
：過分極、再分極
脳血流の自己調節
autoregulation
300
血流量
200
(ml/min)
100
0
0
100
潅流圧（動静脈圧差
200
(mmHg)
-50
単一コンダクタンス
(mV)
10 pS
20 pS
-60
40 pS
静止膜電位
60 pS
100 pS
-70
150 pS
-80
E K = -90
0
20
40
60
80
100
単一細胞で開いているＫチャネルの数
BK Ca , IK Ca ：Ｃａ活性化Ｋチャネル
K ATP
：ATP感受性Ｋチャネル
：電位依存性Ｋチャネル
約100 pS, 約60 pS
約100 pS, 約10 pS
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+channel opener inhibits Ca release from SR
by a thromboxane A2 analogue
0 Ca
0.6
Fura-2
ratio
0.5
3
Force
(mN)
0
U46619
Control
Caffeine
-5
Cromakalim 10 M
10 min
Ca sensitivity and the concentration of KCl (Vm)
Force (%)
100
80
60
Decrease in CaCl2 7
Decrease in KCl
1Ca
Verapamil
6.5
-7
-5
(10 - 10 M)
60K
45K
6
40
90K-2.5Ca
0.3Ca
20
30K
5.5
5
0
0.1Ca
0.03Ca 5K
-20
0
20K
15K
20 40 60 80 100
[Ca2+] i (%)
Relaxation mechanisms of K+ channel openers
+
K channel openers
Agonists
2+
Ca channel
Receptor
Hyperpolarization
Hyperpolarization
Gq
K
GTP
+
2+
[Ca ] i
PLC
GDP
K ATP
DG
+
K
IP 3 <PKC ／rho>
Hyperpolarization
2+
Ca
SR
Force
Ca
2+
sensitivity
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 smoothmuscle 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. 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 M 17bestradiol to the external side of the
membrane.