東北大学機関リポジトリTOUR

Molecular pharmacology of receptors & ion channels

著者 柳澤 輝行

Basic medicine III (Master course)

20120531

Molecular pharmacology of receptors

&

ion channels

• Introduction, Process of signal transduction

• GPCR

Figs. from『新薬理学入門』

– Adrenaline receptors

– Vascular smooth muscle contraction

• Ion channels

– K

+

_{, Ca}

2+

_channels

– Receptor-ion channel interaction

– K

+

_{channel opener}

– Hyperpolarization-relaxation coupling

Teruyuki YANAGISAWA, MD, PhD

The concept & Process of signal transduction

（図２−２）

Phosphorylation

/Dephosphorylation

Transcription

Translation

cAMP

cGMP

IP

3

/Ca

Proteins

Network

Selectivity High affinity Signaling molecules Receptors

Intracellular signaling system

Cellular

response

Transduction Amplification 2nd _Messengers Cascade reaction Changes in Protein Structure & Function

GPCRs as an illustration of F. Jacob’s idea ‘evolution is molecular

le bricolage

evolutionary success of GPCR

Bockaert J and Pin JP: The EMBO Journal Vol. 18,

pp. 1723-1729, 1999

(A) GPCRs have a central common core made of

seven transmembrane helices (TM-I to -VII)

connected by three intracellular (i1, i2, i3) and three

extracellular (e1, e2, e3) loops. The diversity of

messages which activate those receptors is an

illustration of their evolutionary success.

Transmembrane topology of a typical serpentine

receptor (GPCR)

Top view 1 2 3 4 5 6 7 β₂ AR & Adrenaline

Illustration of the central core

of rhodopsin-like GPCR.

The core is viewed from the cytoplasm.

G-Protein α/βγ C

G proteins＝transducers（図2-6）

Small G-Protein (rho)

Ion channels

MAP kinase (byβγ) cAMP IP₃/Ca Prostaglandins Drug-Receptor complex (inactive) (active) Effectors Adenylyl cyclase Phospholipase C Phospholipase A

cholera toxin: Gs , ADP ribosylation. Excess cAMP, diarrhea pertussis toxin: Gi, ADP ribosylation. Inhibition of transduction

RECEPTOR

TARGET ACTION

GIRKs (Kir3.x) Open

N-type Ca channels Inhibit

phospholipase Cb2 Activate G-protein-coupled receptor kinases Adenylyl cyclases PI 3-kinase Several protein

tyrosine kinases Activate

Activate Activate Activate ( MAPK) or

Roles of G

bg

Subunits

G

bg

Hille 3rd p220

β₃W64R

abbreviation

AC: adenylyl cyclase

AR: adrenergic receptor

CA: catecholamine

GIRK: G-protein-activated inwardly

rectifying potassium channel; e.g. K

_ACh

GPCR: G-protein-coupled receptor

MAPK: mitogen-activated protein kinase

N-type Ca

2+

_{↓: N-type Ca}

2+

_{channel inhibition}

PLA

₂

, PLC, phospholipases A

₂

, C

Phylogeny of catecholamine receptors

M3 M4 祖先アミン受容体 M2 M1_M5 b₃ b T b 1 b 2  1A _1B  1D _2C _2B _2A 3 D D 2 D4 1 D D5 Gq Gi/o Gs Gq Gi/o

Ancient amine receptor

Vascular Biologyナビゲ－タ2001, 136-139（改変） COLUMN 2

Molecular signal mechanisms of

contraction

of vascular smooth muscle

『新薬理学入門 第3版』 p.51 図２－２４

Abbreviation

VOC: voltage-dependent Ca

2+

_{channel (Cav)}

ROC: receptor-dependent Ca

2+

_channel

PKC: protein kinase C

RhoK: rho (small G protein) kinase

MLCK: myosin light chain kinase

DG: diacylglycerol

IP

₃

: inositol 1,4,5-trisphosphate

ion channels

•Introduction

•Evolution

•Ca

2 +

_channels

Ca antagonist (Ca channel blocker)

•K

+

_channels

K

+

_{channel openers}

membrane transport system

ion channel

channel

water channel

passive

uniporter

transporter

symporter

antiporter

pump

active

0 m V

- 8 5 m V ≒ E

_K N a + 2 0 n A K+ a b 0.5 nA c C a 2 + 2 n A

Ionic currents & cardiac A.P.

Concentration (mM)

Ouｔ In Ve (mV) Effect on Vm 145 10 +70 Depolarization

2 0.0001 +120 Depolarization

4 150 -94 Hyperpolarization

Ve: equilibration potential 図２－５

Possible Origins of Channel Families

Presumed evolutionary descent of modem ion channels in

animals. Several families can be traced directly to

prokaryotic ancestors, but for some, the ancestral proteins

are not identified. The descendants of K channels are

distinguished according to the number of transmembrane

(TM) segments. Abbreviations: ABC, ATP-binding-cassette

transporters; PPBP, periplasmic binding proteins. Hille 3rd

Ed., Fig. 22.13

Nothing in biology makes sense except in light of evolution. (Dobzhansky)

The VGL-Chanome; 7 families;

143 members

Molecular phylogenity of Cav channel 表２-１

α1 subunit homology(%) (CLUSTAL W)

100 80 60 40 20 0 High voltage activated

Low voltage activated

L（C） L（D） L（F） L（S） P/Q（A） N（B） R（E） T（G） T（H） T（I） C. eleglans C54 DHP-sensitive DHP-nonsensitive Kv channel (gene duplication 2 times) Cav channel Nav channel Nonselective cation channel

L type Cav

channel表２-１



₂

g



b

outside inside H 2N S-S II _III I COOH COOH NH 2 HOOC NH 2 NH 2 COOH



IV

₁

Varadi (1999)

Modulation by

receptor

stimulation

Ca2+

Ca channel disease

C1

⇆ C2 ⇆ O

Sympathetic n.

β-AR

Gs, cAMP

phosphorylation

倉智

CNS, nerve terminus

Inhibitory neurotransmitter

（

_α

₂

_-AR）

‘Willing’

Closed Inactivated Extracellular Intracellular G_bg

‘Reluctant’

Closed Closed Closed Closed Closed Open G_bg

State dependence

of

G

bg

modulation

of

presynaptic Ca

2+

channels

Prepulse deinhibition of

N-type Ca

2+

_channel

Gβ1

PP:prepulse

I(+PP)/I(-PP) 3 2 1 0 τ(-PP)/ τ(+PP) 3 2 1 0 4

7.11

Synaptic Sensitization and Depression

N

C

M1 M2

Kir3.x regulated by

Gi/o βγ

Kir3.x open

hyperpolarization

bradycardia

(K

_ACh

channel)

inhibition of transmission

analgesia

sedation

The historical view. Potassium channels, key controllers of resting and action potentials (A)

Transporters 、

K

+

_recycling

Gastric parietal cell, HCl secretion

K

+

_{channel, p.192 (図7-1)}

Renal tubular cell, Na-K-2Cl, K

+

_recycling

K

+

_{channel, p.175 (図5-6)}

Bartter’s syndrome (antenatal Bartter syndrome, hyperprostaglandin E syndrome) is an electrolyte disorder that has now been recognized to be caused by mutations in at least three transport proteins responsible for NaCl absorption in the loop of Henle. Besides mutations in the Na+/K+/2Cl-cotransporter, Bartter’s syndrome can also be caused by mutations in the K channel that is present in the apical membrane of the ascending limb (ROMK or KIR1.1). This K channel is a K recycling pathway and its operation is a prerequisite for NaCl absorption through NKCC2. Clinically, Bartter syndromes types I and II are indistinguishable. In contrast, a milder form of Bartter’s syndrome is caused by mutations in the basolateral chloride channel (ClC-Kb), an exit pathway for cellular Cl. →Diuretics

Vm （mV） －55 －90 K ＋ channel Ca 2＋ channel Vascular sm.m.tone Vascular diameter K＋ channel opener hyperpolarization K＋ －75 －40 rested Ca 2＋ depolarization K ＋ open Hyperpolarization-relaxation coupling K ＋ K ＋ K ＋ K ＋ K ＋ Ca 2＋ Ca 2＋ Ca 2＋ Ca 2＋ Ca 2＋ close close open Hypertension, spasm K+ channel in Vasc. sm. m. 図４－９

K _ATP BK , IK_{Ca Ca} Kv 約100 pS, 約10 pS 約10 pS 約100 pS, 約60 pS -90 -80 -70 -60 -50

Vm (mV)

100 80 60 40 20 0 150 pS 10 pS 20 pS 40 pS 60 pS 100 pS

Single channel conductance

Open channel number in vascular sm. m. cell E _{K =}

柳澤輝行：カリウムチャネルと 過分極弛緩連関．総説 創薬

科学_{−分子から個体への薬理}

Blood flow

autoregulation

0

₁₀₀

₂₀₀

Mean Blood Pressure(mmHg)

(ml/min)

0

100

200

300

b. Pulmonary circulation Hypoxia BKCa,IKCa Kv and/or KCa,ATP a. Systemic circulation K _ATP Kv Hypoxia, Ischemia, adenosine

Ｋ + channel opener

Blood pressure ↑

（Stretch-activated channel）

Regulation of arterial sm. m. tone

COLUMN 5 p52

BKCa,IKCa

Autoregulation Myogenic tone

Depol.

Ca2+ _influx

through Cav channel

[Ca2+_] i↑

Depol.

Ca2+ _influx

through Cav channel

[Ca2+_] i↑ Hypoxic pulmonary vasoconstriction Hyperpolarization, repolarization close close open

Hypoxic pulmonary vasoconstriction

ventilation-perfusion ratio inequality

A B

Hypoxic (A)

Normoxic (B)

air

Pulmonary artery A B

Hypoxemia

PaO

₂

↓

intrapulmonary shunt

↓

Maintain PaO

₂

(short term)

Pulmonary hypertension

(long term)

alveolus

Pulmonary vein

2 2 2 O O O K channel I ICa

carotid sinus nerve

P

O

2

sensor in carotid body

生理学テキスト

図16-26 PO2 normal PO2 low INa capillary CNS

Oxygen Sensing: It’s a Gas!

Modified from SCIENCE VOL 306 17 DECEMBER 2004 p2050

K

+

hyperpolarization

Guanylyl cyclase

Mechanisms of insulin secretion in β cell

図９

_-３

glucose Ca 2+ Gi A kinase insulin Gs AC PLC Gq IP3 Ca 2+ cAMP ATP glibenclamide +

-

+ P Depol.

K

_ATP

-

ATP ACh [K ] + 0 ↑ + + Ca store (ER) SU R + +

-

L type (M 3 )

b

₂



₂ GLP-1

Adrenaline

[L cell [D cell] diazoxide + GLUT2 somatostatin

minoxidil, LP 805 Pyrimidine: O N N _{N H} 2 H₂N N trichogen Benzopyran: Cromakalim O H N C O C H ₃ C H ₃ N O * _*

K

+

channel openers

Pyridine:

nicorandil

KRN2391 C O N H C H ₂C H ₂O N O ₂ N "N-K hybrid" NO, cGMP ↑ Hyperglycemia (side effect) Insulin secretion↓ K_ATP open in β cell

3 N NH S O Cl CH 2 Benzothiadiazine: diazoxide "Nonspecific KCO" Hypertricosis as a side effect

cf. ischemic preconditioning;

pharmacological

C o n t r o l

F

u r a - 2

r

a t i o

0

. 6

0

. 5

F

o r c e

(

m N )

0

3

1 0 m i n

U 4 6 6 1 9

C a f f e i n e

C

r

o

m

a

k

a

l

i

m

1

0

-5

M

0

C a

Naunyn-Schmied. Arch. Pharmacol. 1992;346:691-700. Biochem. Biophys. Res. Commun. 1992;187:1517-22. K+ _{channel opener inhibits IP}

3 generation & Ca2+ release from SR by a thromboxane A₂ analogue. Hyperpolarization-relaxation coupling

Molecular signal mechanisms of

contraction

of vascular smooth muscle

『新薬理学入門 第3版』 p.51 図２－２４

Noradrenaline Serotonin

Canine

basilar artery

loaded with

fura-2

(a

[Ca

2+

_]

i

indicator).

Serotonin Iberiotoxin Force (mN) 0 5.0 0.66 0.82 Levcromakalim Ratio (F340/ F380) 90 mM KCl 10 min

Fundam. Clin. Pharmacol. 1998;12:403-10. 90mM KCl makes depolarization (Vm≒－10mV, 60Xlog[90/150])

Influences of K

+

_{channel blockers on}

levcromakalim-induced reduction of [Ca

2+

_]

i

and contractile force

0 20 40 60 80 100 120 140 160 180 Levcromakalim

Glibenclamide IbTX TEA Control [Ca ] i or Force (%) 2+

0 20 40 60 80 [Ca2+ ]_o (mM) F o rc e (% )

b

-20 100 0 0.1 0.3 1 2.5 10 ** ** ** * * * * [C a ] i (% ) 2+

a

-60 -40 -20 0 20 40 60 80 100 * * * * Influences of changing extracellular Ca2+ concentration ([Ca2+_] o) on [Ca2+]i and force in the

presence (circle) or absence (square) of serotonin. Levcromakalim (closed) was applied 5 min before the application of serotonin.

Relationship

between [Ca

2+

_]

i

and force of

contraction

induced by

serotonin ( 10

-6.5

M) in the absence

(

○

) and presence

of levcromakalim

( 10

-5.5

_M,

_●

_{) or}

nicardipine (

■

).

-60 -40 -20 0 20 40 60 80 0 10 20 30 40 50 60 70 80 90

[Ca

2+

]

_i

(%)

c

-10 100

F

o

rc

e

(%

)

0 0.1 0.3 1.0 2.5 10 0 0.1 0.3 1.0 2.5 10 *** 10-6.5 _M 10-5.5 _M

Ca sensitivity ↓

KCl depolarization revisited.

Yanagisawa T & Okada Y: Am J Physiol 1994;267:H614-621.

KCl depolarization revisit.

Am J Physiol 1994;267: H614-621.

Eliminating Ca

2+

_{influx by 2 ways}

Effects of eliminating Ca2+ _{influx by repolarization (○) or removal of} extracellular Ca2+ _{concentration ( 0 Ca, [Ca}2+_]

o;●) on increased intracellular Ca2+ _{concentration ( [Ca}2+_]

i) and force in depolarized canine coronary arterial muscles loaded with fura 2.

Repolarization

0 Ca

0

2

0

4

0

6

0

8

0

1

0 0

F

o

r c

e

( %

)

[

C a ] ( % )

2

+

_i

1

0 0

8

0

4

0

0

2 0

6

0

-

2 0

6

5

7

6 . 5

5 . 5

9

0 K - 2 . 5 C a

1

C a

0

. 3 C a

0

. 1 C a

0

. 0 3 C a 5 K

6

0 K

4

5 K

3

0 K

2

0 K

1

5 K

D

e c r e a s e i n K C l

D

e c r e a s e i n C a C l

V

e r a p a m i l

(

1 0 - 1 0 M )

2

- 7

- 5

Ca sensitivity and the concentration of KCl (Vm)

KCl depolarization as a calcium-sensitizing stimulus.

modified from Ratz PH et al. AJP 2005;288:C769-C783.

Ca sensitivity ↑

Relaxation mechanisms of K

+

_{channel openers}

DG GTP GDP Receptor <PKC ／rho> Ca sensitivity2+ Ca channel2+ K+ K+

K channel openers

+

PLC G

Force

2+ i

[Ca ]

Ca2+ SR KATP IP₃ Hyperpolarization _{Hyperpolarization} Agonists Hyperpolarization q

Summary

• Molecular mechanisms of intracellular signaling

• Interaction of receptors & ion channels

• K

+

_{& Ca}

2+

_{channels in vascular smooth muscle}

– Agonist-induced contraction

– Autoregulation of blood flow

– Hypoxic pulmonary vasoconstriction

– K

+

_{channel openers; Nicorandil (NK hybrid)}

教科書, 参考図書, 文献

TOUR 東北大学機関リポジトリ

柳澤輝行編著：新薬理学入門3版（2008）

カッツング薬理学（原著10版） （2008）

大地陸男：生理学テキスト6版（2010）

Hille, 3

rd

Ed. (2001)

Kandel 4

th

Ed. (2000)

唐木英明 編著：イオンシグナルの謎

_{−カルシウ}

ムの40億年を渉猟する(1999)

倉智嘉久：心筋細胞イオンチャネル(2000)

設問（４）

_{Please explain the result obtained from canine}

coronary arterial smooth muscle loaded with fura-2 (a

[Ca

2+

_]

i

indicator). U46619: thromboxane A

2

analogue

基礎医学３

20120531

イオンチャネルと受容体の分子薬理学

Pharmacology of ion channels & receptors

• 情報伝達signal transductionの基本課程

• Gタンパク質共役型受容体GPCR

• カテコールアミン受容体タイプ

の

分類

と

性質

catecholamine receptors

• 膜輸送タンパク質transporters の分類

• イオンチャネル ionic channels

•

イオンチャネル・受容体相互作用receptor-ion channel interactイオンチャネル・受容体相互作用receptor-ion