生物醫學特論

„ Cell, cell membrane and membrane proteins

Methods of studying ion channels

Ca2+ channels and diseases „

„ Classification and function of ion channels

陳建璋 „ Channelopathies
中央研究院生物醫學科學研究所
ccchen@ibms.sinica.edu.tw
2652-3522
1 2

9/18/2006

Mouse ventricular myocytes

小鼠心室細胞

50 µm

3 4
http://www.kottke.org/plus/photos/200105europe/castle.jpg
http://www.cytochemistry.net/Cell-biology/membrane_intro.htm
 Na+/K+ Pump

[Na+]o=145 mM [K+]o=5 mM

++++

-- --

[Na+]i=5 mM [K+]i=140 mM

Molecular Biology of the Cell

5 6
Molecular Biology of the Cell

How to study ion channel?

„ Cell, cell membrane and membrane proteins - +

„ Methods of studying ion channels

„ Classification and function of ion channels

„ Channelopathies
- +
-
+

Ohm’s Law: V =IR 電壓＝電流 X 電阻

7 8

http://opal.msu.montana.edu/cftr/IonChannelPrimers/beginners.htm
 Voltage-clamp technique
二十世紀中葉（1950年代）：
霍去金 (Hodgkin) &赫胥黎 (Huxley)研究烏賊的巨大神經元軸突
→發現神經細胞內外具有電位差（神經細胞內的電位較低≒－65mV）
(1)無脊椎動物（烏賊、龍蝦、蚯蚓）具有巨大的神經元
(2)烏賊的神經元軸突直徑＞1mm
可用來測量靜止膜電位的伏特數（烏賊的靜止膜電位≒－65mV）
可用來記錄在神經衝動傳遞時，離子流動所造成的電位變化
(烏賊的動作電位≒＋40mV）

Nobel Prize in 1963

Hodgkin Huxley 9 10

Voltage-clamp methodology of Hodgkin and Huxley Action potential and Na+ and K+ currents
recorded from squid axon

Neuroscience by Purves et al. 最早的細胞內紀錄-動作電位

11
1939 Hodgkin and Huxley 12
 How to record a single cell?
如何記錄單一細胞？ Patch-Clamp Technique

Smooth muscle cells

奈爾 (Neher) 與沙克曼 (Sakmann)
(diameter <10 mm)
平滑肌細胞

Mouse ventricular myocytes

小鼠心室細胞

Nobel Prize in 1991

50 µm
13 14

Patch-Clamp Technique
電壓＝電流 X 電阻
„ Tight seal onto the
membrane – GΩ seal
„ Ion channel is
trapped under the
pipette. Pipette tip
1-2 µM
„ Pipette is connected
to an amplifier for
voltage control.
„ Measure channel
activity
Neuroscience by Purves et al.
15 16
http://www.cellsalive.com/patch.htm
Cell-attached
recording

Na+ channels : comparison of single channel with whole cell

17 18
Variations of the patch clamp technique. recordings

How does the channel sense change in voltage?

„ Positively charged residues within the S4 TM segment.

„ Evidence that the segment rotates out of the membrane in
response to change in voltage: gating charges

19 20
 The third Nobel Prize for ion channel research Aquaporin-水通道

Agre MacKinnon
阿格雷 麥金農

Nobel Prize in 2003

21 22

Aquaporin-水通道
K+ channel-鉀離子通道

Fig 3. Passage of water molecules through the aquaporin AQP1.

Because of the positive charge at the center of the channel, positively
charged ions such as H3O+, are deflected. This prevents proton leakage 23 24
through the channel.
 „ Cell, cell membrane and membrane proteins

„ Methods of studying ion channels

„ Classification and function of ion channels

„ Channelopathies

25 26
Jiang et al., Nature (2003)

Types of ion channels

Types of ion channels
離子通道的種類
Voltage-gated Ligand-gated Mechanical-gated
電壓驅動式 配體驅動式 機械驅動式 Voltage-gated 配體驅動式離子通道

Na+ K+ Ca2+ acetylcholine glutamate glycine

鈉離子 鉀離子 鈣離子 乙烯膽鹼 谷氨酸鹽 甘氨酸

可興奮性細胞 Excitable neuron

神經細胞 興奮性神經細胞
骨骼肌細胞 Inhibitory neuron
心肌細胞 抑制性神經細胞
平滑肌細胞
27 28

Molecular Biology of the Cell

 Classification of voltage-gated Ca2+ channel Subunit Composition of High Voltage-Gated Ca2+ channel

α2
γ α1

δ
L-type

β
29 30

Functions of Ca2+ channels

Voltage-gated Ca2+ channel α1 subunit
Ligand-gated Voltage-gated
Ion Channels Calcium Channels

I II III IV

+ + + +
+ + + +
S1S2S3S4S5 S6
+ + + +
+ + + +

Muscle Pacemaking
contraction
Ca2+ activity

Neurotransmitter Gene
release expression

31 32
 Neuromuscular junction
Nerve
terminal
Muscle
cell
Ion Channelopathies
離子通道相關疾病
Cystic fibrosis (Cl- channel; CFTR)
Thomsen Myotonia congenita(Cl- channel; CLC-1)
Becker Myotonia congenita (Cl- channel; CLC-1)
Hypercalciuric nephrolithiasis (Cl- channel; CLC-5)
Bartter's syndrome type 1 (Cl- channel; CLC-Kb)
Angleman / Prader-Willi (GABA channel; GABAAB3)
Low molecular weight proteinuria (CLCN5)
Bartter’s syndrome type 3 (SLC12A3)
Startle disease (hyperexplexia) (glycine receptor)
Liddle's syndrome (ENaC; SCNN1A, SCNN1B)
Paramyotonia congenita (Na+ channel; SCN4A)
Hyperkalemic periodic paralysis (Na+ channel; SCN4A)
Myotonia Fluctuans (Na+ channel; SCN4A)
Myotonia Permanens (Na+ channel; SCN4A)
Acetzolamide-responsive myotonia (Na+ channel; SCN4A)
Malignant hyperthermia (Na+ channel; SCN4A)
Idiopathic ventricular fibrillation (Na+ channel; SCN5A)
Long QT Syndrome (LQT3 Na+ channel; SCN5A)
Epilepsy with febrile seizures (Na+ channel; SCN1B immune)
Acute motor axonal neuropathy (Na+ channel; immune)
Guillain-Barré & CIDP (Na+ channel; immune)
Multifocal Motor Neuropathy (Na+ channel; immune)
Nephrogenic diabetes incipidus (AQP-2)
Total color blindness (CNGA3)
„ Cell, cell membrane and membrane proteins
„ Methods of studying ion channels
„ Classification and function of ion channels
„ Channelopathies
Ach receptor
VGCC
VGSC
33 34
Calcium Channelopathies
鈣離子通道相關疾病
Hypokalemic periodic paralysis (Ca2+ channel; CACNL1A3) „ 低血鉀週期性無力症 Hypokalemic periodic paralysis (α1S)
Malignant Hyperthermia (Ca2+ channel; CACNL1A3, RyR 1)
X-linked congenital night blindness (Ca2+ channel; CSNB2) „ 惡性高熱綜合症 Malignant Hyperthermia (α1S,RyR 1)
Muscular dysgenesis (rodent Ca2+ channel; CACNL1A3)
Episodic ataxia type-2 (Ca2+ channel; CACNL1A4)
„ 先天性夜盲症 X-linked congenital night blindness (α1F)
Familial hemiplegic migraine (Ca2+ channel; CACNL1A4)
Spinocerebellar ataxia (Ca2+ channel; CACNL1A4)
„ 肌肉萎縮症 Muscular dysgenesis (α1S)
Congenital myasthenic syndrome (nAChR) „ 陣發性運動失調症 Episodic ataxia type-2 (α1A)
Lambert-Eaton Myasthenic Syndrome (Ca2+ channel; immune)
Insulin-Dependent Diabetes (Ca2+ channel; immune) „ 偏頭痛 Familial hemiplegic migraine (α1A)
Antenatal Bartter’s Syndrome type 2 (K+ channel; KCNJ1)
Long QT Syndrome (LQT1; K + channel; KCNA8) „ 脊髓小腦萎縮症 Spinocerebellar ataxia (α1A)
Long QT Syndrome (LQT2 K+ channel; HERG)
Jervell & Lange-Nielsen Syndrome (K+ channel; KCNE1, KCNQ1) „ 癌肌症 Lambert-Eaton Myasthenic Syndrome (auto-immune, α1A)
Episodic Ataxia / Myokymia Syndrome (K+ channel; KCNA1)
Benign neonatal epilepsy (K+ channel; KCNQ2, KCNQ3)
„ 糖尿病 Diabetes (α1C, α1D Ca2+ channel; immune)
Schizophrenia (K+ channel; KCNN3)
Retinitis pigmentosa (KNS channel; CNGA1)
„ Timothy syndrome (α1C)
Rod monochromacy (KNS channel; CNGA3) „ Autism (α1C, α1H)
Hyperinsulinism of Infancy (K+ channel; SUR1)
Hyperinsulinism of Infancy (K+ channel; Kir6.2) „ Childhood absence epilepsy (α1H)
Visceroatrial Heterotaxia (gap junction; CXA1)
CMT-X (gap junction; CXB1)
Non-syndromic deafness (gap junction; CXB2) 35 36
 鈣離子通道與疾病 Hypokalemic periodic paralysis(HypoPP)
(低血鉀週期性無力症)

P/Q-type „ An autosomal dominant inherited disorder that causes occasional episodes

N-type of muscle weakness.
Nervous system Nervous system „ Onset usually begin in adolescence, but they can occur before age 10 and
epilepsy pain has reduced penetrance in females.
migraine anxiety „ Attacks can be provoked by rest after exercise and factors that lower
migraine serum K levels. It usually last between a few hours and one day.
„ Three mutations in the α1S gene (chromosome 1).
„ It is not obvious how altered calcium currents would produce persistent
depolarization triggered by low K.
L-type
Heart HypoPP
T-type
hypertension Heart, Nervous system MH
arrhythmias epilepsy Muscular dysgenesis mouse
angina cardiovascular disease

37 38

(Eurekah Bioscience1:2, 125, 2005)

Malignant hyperthermia (MH) Incomplete X linked congential stationary night blindness

(惡性高熱綜合症)
„ An autosomal dominant inherited disease that causes a rapid rise in body
temperature (fever) and severe muscle contractions when the affected
person undergoes general anesthesia. The anesthetized patient quickly
develops muscle rigidity. Body temperature rapidly rises to 40.5 0C or higher.
During these episodes, muscle tissue is destroyed and, eventually, kidney
failure may result and could be lethal.
„ About 29 mutations found in ryanodine receptor (RyR1, chromosome 19)
gene, 1 in α1S gene.
„ RyRs control the Ca release from SR. MH mutation cause massive Ca release
triggered by halogenated anesthetics.
(CSNB2)
„ CSNB2 is a non progressive, dominantly inherited disorder.
„ Symptoms include night blindness, decreased visual acuity, myopia,
nystagmus, strabismus and abnormal ERG results.
„ Impairments resulted from alter synaptic transmission from
photoreceptor cells to 2nd order neurons.
„ 30 Mutations in the α1F L-type voltage gated calcium channel (VGCC)
(Cav1.4)

39 40

(Eurekah Bioscience1:2, 125, 2005)

 Tissue distribution of the Cav1.4 calcium channel The Lambert-Eaton myasthenic syndrome (LEMS)
(癌肌症 )

„ Lambert-
Lambert-Eaton myasthenic syndrome (LEMS) is a rare condition in
which weakness results from an abnormality of acetylcholine (ACh
(ACh))
release at the neuromuscular junction.
„ ~40% of small cell lung cancer (SCLC) patients with LEMS
„ LEMS results from an autoimmune attack against P/Q type voltage-
voltage-
gated calcium channels (VGCC) on the presynaptic motor nerve
terminal.
„ Prednisone, plasma exchange (PEX) are effective treatments.

41 42

J Neurosci, 24(7), 1707, 2004

Mutations in α1A cause ataxia and epilepsy Episodic Ataxia type 2 (EA2) (運動失調症 )

„ EA2 is a dominantly inherited human disorder with symptoms

including ataxia, nystagmus (眼振), dysarthria (口齒不清) and
vertigo (眩暈). Cerebellar atrophy is common and 50% of EA 2 patients
report migraine like symptoms.
„ induced by stress, exercise and lasts hours to days.
„ Onset time: late childhood or adolescence.
„ 16 mutations in α1A VGCC (chromosome 19).
Most of them cause reduction of Ca current.
„ The big intra familial cariability in penetrance and symptoms suggests
that environmental, hormonal and/or genetic factors other than the a1A
mutation are important for the phenotype of EA2 and SCA6.

Episodic ataxia type 2

Episodic and progressive ataxia
Generalized epilepsy and epsodic and progressive ataxia
43 44
Spinocerebellar ataxia 6
 Spinocerebellar Ataxia 6 (SCA6)
(遺傳性小腦脊髓運動失調症 )
Familial hemiplegic migraine (FHM) (偏頭痛)

„ SCA 6 is a dominantly inherited human disorder with symptoms such as
ataxia, nystagmus, dysarthria and neuronal loss in
the cerebellum and the dentate and the inferior olivary nuclei.
„ Late onset (40-50 years of age)
„ Expanded CAG repeat in exon 47 of α1A gene.
„ The big intra-familial variability in penetrance and symptoms suggests
that environmental, hormonal and/or genetic factors other than the α1A
mutation are important for the phenotype of EA2 and SCA6.
„ FHM is a dominantly inherited human disorder with symptoms
including headaches accompanied by aura and hemiparesis, ataxia and
nystagmus.
„ Onset time: childhood or adolescence
„ 14 mutations in α1A VGCC. Most of them cause reduction of Ca current.

45 46

Timothy Syndrome

„ Timothy syndrome, a novel disorder characterized by multiorgan dysfunction

including lethal arrhythmias, webbing of fingers and toes, congenital heart
disease, immune deficiency, intermittent hypoglycemia, cognitive
abnormalities, and autism.
„ In every case (17), Timothy syndrome results from the identical, de novo
CaV1.2 missense mutation G406R.
CaV1.2 missense mutation G406R
Leads nearly complete loss of
voltage-dependent channel
inactivation. This likely induces
intracellular Ca overload.

47 48
Cell. 2004 Oct 1;119(1):19-31.
Mutations identified in individuals with ASD alter the activation Childhood Absence Epilepsy (兒童癲癇症候群)
properties of Cav3.2 channels
„ Annual incidence has been estimated at 1-8 per 100,000 in children aged
0-15 years, comprising 8% of epilepsy cases in school-aged children.

„ Mutations in α1H cause greater calcium influx during physiological

activation

Locations of mutations or single nucleotide polymorphisms found in CACNA1H

49
Neurosci Lett. 2006 Oct 2;406(1-2):27-32 50
Ann Neurol. 2003 Aug;54(2):239-43
J. Biol. Chem. 2006;281:22085-22091

Idiopathic generalized epilepsy (體質性汎發性癲癇 ) Potential mechanisms

Genetic variation of CACNA1H in idiopathic generalized epilepsy Mutations in calcium channels

Ann Neurol 2004; 55: 595-596 Altered Channel kinetics/gating
Altered activity modulation
Altered channel expression/turnover
Coding and noncoding variation of the human calcium-channel Altered membrane targeting

beta4-subunit gene CACNB4 in patients with idiopathic generalized

epilepsy and episodic ataxia. Altered calcium entry
Am J Hum Genet. 2000 May;66(5):1531-9 Altered synaptic transmission Altered intracellular calcium level

Synaptic changes lasting seconds to minutes

Changes in activities of
modulatory proteins
kinases/phosphotases lasting
minutes to hours
Episodic ataxia
hemiplegic migraine
Progressive ataxia Altered gene expression/
Developmental changes with
Epilepsy
long lasting effects

51 52
 Tottering (tg), Leaner, Rolling Nagoya and Mutations in auxiliary subunits of Ca2+ channels
Rocker

„ Tottering is a recessively inherited neurological disease of mice with

seizures (absence and focal motor) and mild ataxia.
„ Single nucleotide mutation in calcium channel a1A (Cacna1A). This α2
lead to a reduction of calcium current by 50%.
γ α1
„ Learner is also a recessively inherited neurological disease of mice with
sever ataxia and a substantial loss of cerebellar Purkinje and granule δ
cells.
„ Single nucleotide mutation in calcium channel α1A (Cacna1A).

„ Rolling Nagoya is recessive and exhibit poor motor coordination of

hindlimbs and sometimes stiffness of hindlimbs and tail, but they lack
apparent seizures.

„ Rocker exhibit ataxia, unstable gait accompanied by intention tremor,

typical of cerebellar dysfunction. β
53 54

Ducky Mouse (du) Lethargic mice (lh)

„ Lethargic mice exhibit ataxia, spontaneous focal motor seizure and

„ Spontaneous mutation show a waddling or reeling gait and a
tendency to fall to one side.
„ Homozygous mutant mice are slightly smaller than normal and
may occasionally have seizures.
„ Severe dysgenesis of hindbrain and spinal cord, myelin
deficiency.
„ Mutation in calcium channel α2/δ-2 (Cacna2d2)
absence seizure, but without apparent neuronal degeneration.
„ 4 nucleotides insertion into β4. This causes exon skipping and
the generation of transcripts with a premature stop codon. Subsequent
studies have demonstrated that there is no detectable protein, indicating
that this is indeed a null mutation.

55 56
 Abnormal Vocalization of α1H-/- Mice

57 58

Abnormal Trachea Formation

+/+ +/- -/- +/+ -/-

+/+ -/- E16 +/+ -/-

c c c

P1 P1 E15.5 E15.5 59