CIPA project
Protocols of cardiac ion channels
hERG, Ca++ and Na+
Eunjung Park
FDA/CDER/OND/DCRP
herg
1
Summary of protocols
hERG
hCav1.2
hNav1.5
Literature sources
Chantest, Merck, Abbott,
GSK, AZ, UW and other
academic articles (18
papers for a summary)
Chantest, Merck, Abbott,
AZ, Aventis, and other
academic articles (11
papers for a summary)
Chantest, Merck, GSK,
Aventis, AZ, UW, and
other academic articles
(17 papers for a
summary)
Period of literatures
1997-2013
2003-2013
1998-2013
Manual
HEK293 (12) > CHO (3) >
myocyte (1) > mouse Lcell (1)=SH-SY5Y (1)
Myocytes (4)> HEK293 (2)
> HL-1 (1)
HEK293 (10) > CHO (2)>
myocyte (2)
Automatic
CHO (6) > HEK293 (2)
CHO (4)> HEK293 (1) >
myocyte (1)
CHO (3) = HEK 293 (3) >
myocyte (1)
Manual
Near Physiological (8) >
Room temperature (6)
RT
RT
Automatic
RT
RT
RT
Manual
Whole-cell patch clamp
Whole cell parch clamp
Whole cell parch clamp
Automatic
Whole cell patch clamp
or perforated patch clamp
Whole cell patch clamp
or perforated patch clamp
Whole cell patch clamp
or perforated patch clamp
Cells
Temp
Recording
summary
2
Access and seal resistance of manual patch clamp assays
 Pipette (tip) resistance (access resistance, electrode resistance):
•
< 5M in 67 out of 70 literatures (96%) including hERG, Ca++ and Na+ channels
pipette resistance (M)
6.0
5.0
4.0
*
*
3.0
*
2.0
1.0
0.0
1 2 3 4 5 6 7 8 9 10111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758596061626364656667
literature
 Seal resistance:
• 3 (* on graph) out of 70 protocols (4.3%) indicated its seal resistance as > 1G.
 Series resistance compensation:
• 35 out of 70 protocols (50%) indicated its series resistance compensation (minimum
60% and maximum 90%).
summary
3
Seal resistance in automatic patch clamp assays
PatchXpress
IonWork
Barracuda
IonWork HT
IonWork
Ref ID (yr)
88 (2009)
92 (2010)
121 (2011)
151 (2005)
61 (2013)
62 (2006)
101 (2008)
Ion channel
Ca++
Na+
hERG/Na+
hERG
hERG
hERG
Na+
Sponsor
Merck
Merck
GSK
J&J
GSK
AZ
AZ
Cell
HEK293
HEK293
HEK293
HEK293
CHO
CHO
CHO
1-3
1-9
(ave 2.4)
1-2.8
(ave 1.7)
> 30
(ave. 58)
> 60
> 60 (HT)
> 30 (Quattro)
amphotericin
amphotericin
Amphotericin
Quattro (Escin)
Re (M)
Rseal (M)
2520
>1000
1083
1900
Rm (M)
1358
>150
>200
>200
(ave 710)
11.8
<15
(ave. <10)
<15
14
suction
suction
suction
suction
Ra (M)
Membrane
rupture
Rseal: Patch seal resistance, Rm: membrane resistance during whole cell recording, Ra: whole cell access resistance, Re: electrode resistance
summary
4
Extracellular buffer
hERG
hCav1.2
hNav1.5
M
A
M
A
M
A
NaCl






KCl





CaCl2*






MgCl2






HEPES






glucose






KH2PO4
()
()
Na2HPO4
()
()
NaHCO3
()
EGTA
()
NMDG
()
CsCl
()
Cholin-Cl
()
TEA-Cl
()
()
BaCl2*
()
()
L-aspartic acid
()
()
()
()
()
M: conventional manual patch clamp (mainly, Axopatch)
A: Automatic patch clamp
(): included in less than half of protocols
summary
5
Intracellular buffer
hERG
Current carrier
K-aspartate
K-gluconate*
Cs
(K block)
CsCl
Cs-aspartate
Cs-methanesulphonate
Fluoride
(Ca block)
KF
NaF
CsF
hCav1.2
M
A

()
hNav1.5
M
A
M
A








()
()
()
KCl*


MgCl2






EGTA






HEPES









()




()
()
()


()
()
GTP
ATP
Na2ATP
K2ATP
MgATP
ATP
regeneration system
Creatine phosphokinase
Creatine phosphate
Tris-phosphoreatine
Seal resistance
TEA (tetraehtylammonium)

()
CaCl2

()
NaCl**
()
()
()
M: conventional manual patch clamp (mainly, Axopatch) A: Automatic patch clamp, (): included in less than half of protocols
summary
6
Representative pulse sequences for a drug evaluation
Depolarizing
(0 to 70 mV, 1-4s)
hERG
Repolarizing
(-80 to 20 mV, 2-6s)
Vh=-80 mV
Depolarizing
(0 to 20mV, 100 to 500 ms)
hCav1.2
Vh=-90 to -40 mV
Depolarizing
(-30 to 0 mV, 10-300 ms)
hNav1.5
Hyperpolarizing
(-120 mV)
Vh=-150 to -80 mV
Ref: JCE 2010, 21, 301
summary
7
hERG assay
herg
8
Representative intracellular buffer solution
Intracellular (pipet) (mM)
Source
Year
Amplifier
K-aspartate
Kgluconate
KCl
pH
pH
buffer
5
7.2
KOH
5
3.2
7.35
KOH
5
5
1
7.3
KOH
5
10
1
7.2
KOH
20
1
1
7.35
KOH
10
1
0.1
7.2
KOH
10
10
1
1
7.2
KOH
70
5
EGTA
ATP
5
15
KF
HEPES
MgCl2
4
10
5
5
20
10
5
CaCl2
NaCl
manual
Chantest
2013
AxoPatch
Merck
2006
AxoPatch
Abbott
2012
AxoPatch
UWM
2006
AxoPatch
130
Automatic
GSK
2013
Barracuda
140
GSK
2013 PatchXpress
GSK
2013
130
119
125
10
5
5
100
10
5
2008 PatchXpress
60
5
AZ
2012
IonWorks
140
1
20
1
7.3
KOH
AZ
2012
IonWorks
40
3
5
3.2
7.3
KOH
Merck
130
QPatch
100
15
7.2
UWM: University of Wisconsin at Madison
herg
9
Representative extracellular buffer solution
Extracellular (superfusion) (mM)
Source
Year
amplifier
NaCl
KCl
CaCl2
MgCl2
HEPES
glucose NaHCO3
pH
pH buffer
Manual
Chantest
2013
AxoPatch
137
4
1.8
1
10
10
7.4
NaOH
Merck
2006
AxoPatch
132
4
1.8
1.2
10
11.1
7.35
NaOH
Abbott
2012
AxoPatch
140
5
2
1
20
5
7.4
NaOH
UWM
2006
AxoPatch
137
4
1.8
1
10
10
7.4
NaOH
GSK
2013
Barracuda
136
3
2
1
20
6
7.35
NaOH
GSK
2013
PatchXpress
140
4
2
1
10
10
7.4
NaOH
GSK
2013
QPatch
145
4
2
1
10
10
7.4
NaOH
Merck
2008
PatchXpress
132
4
3
0.5
10
11
7.35
AZ
2012
IonWorks
137
4
1.8
1
10
10
7.3
Automatic
UWM: University of Wisconsin at Madison
herg
12
NaOH
10
Pulse sequences: potency
Source-Yr
temperature
sample
replication
pulse
holding
(mV)
1st pulse
(time, mV)
2nd pulse
(time, mV)
interval
Chantest-2013
ambient
n/a
step
-80
2s, 40
2s, -40
10s
NDA- 2007
35±2
>3
step-ramp
-80
1s, 20
0.5mV/ms, +20 to -80
5s
Merck-2006
35 ± 0.5
5-8
step
-80
1s, 20
2s, -50
15s
NDA- 2004
35
5-8
step
-80
1s, 20
2s, -50
15s
Abbott-2012*
36.5 -37.0
5
Step-ramp
-80
1.5s, 0
1mV/25ms, 0 to -80
15s
NDA- 2002
36.5 -37.0
6
step
-80
3s, 0
4s, -50
15s
Dr. January-2009
23 ± 1
3-6
step
-80
4s, 70
5.7s, -50
15s
Pfizer/Univ Walk2010
37
n/a
step
-80
2s, 20
4s, -40
12s
37
n/a
Step-ramp
-80
1s, 20
0.5mV/ms, +20 to -80
10s
* hERG enhancer
herg
11
Pulse sequences: Voltage dependent
temperature
holding
(mV)
1st pulse
(time, mV)
2nd pulse
(time, mV)
37
-80
n/a, -60 to 40
n/a. -100
37
-80
3s, -60 to 40
(10mV inc)
4s, -60
HEK 293
35
-80
4s, -60 to 50
5s, -50
January-2005
HEK293
23
-80
4s, -70 to 60
(10 mV/15s)
6s, -50
Hancox-2010
HEK 293
Dofetilide/cisapride
37
-80
2s, -40 to 50
4s, -40
Belardinelli-2008
HEK 293
ranolazine
23
-80
4s, -80 to 70
( 10mV inc.)
5.7s, -50
Fox-2009
Canine
myocytes
37
-80
0.5s, 60 to -30
(10 mV inc)
1.6s, -30
source
cells
Abbott-2009
HEK 293
Abbott-2007
HEK293
January-1998
Drug
chloramine-T
herg
12
Pulse sequences: Channel activation
source
Drug
temperature
holding (mV)
1st pulse
(time, mV)
2nd pulse
(time, mV)
Abbott-2009
A-935142*
37
-80
5-850 ms (30ms inc), -10
n/a, -100
Abbott-2007
chloramine-T
37
-80
5-185 ms, 30
n/a, -100
Abbott-2007
chloramine-T
37
-80
5-480 ms, 0
n/a, -100
January-2005
Miconazole
23
-80
various duration, 20
1s, -50
* hERG enhancer
herg
13
Pulse sequences: Channel deactivation
source
Drug
temperature
holding (mV)
1st pulse
(time, mV)
2nd pulse
(time, mV)
Abbott-2009
A-935142*
37
-80
n/a, 40
n/a, -70 to -120
Merck-2006
Flunarizine
35
-80
1s, 50
2s, -120 to 20
(10mV inc)
January-2005
miconazole
23
-80
1s, 60
5s, -100 to -20
(10 mV inc)
* hERG enhancer
herg
14
Pulse sequences: Channel inactivation
1st pulse
(time, mV)
2nd pulse
(time, mV)
3rd pulse
(time, mV)
-80
500ms, 60
2 ms, -100
n/a, -40 to 60
37
-80
500ms, 60
2 ms, -100
n/a, -20 to 60
miconazole
23
-80
200 ms, 60
100ms, -100
300ms, -20 to 60
HEK 293
ranolazine
23
-80
300 ms, 60
10ms, -100
n/a, -40 to 40
(10 mV inc.)
Pfizer/Univ-2010
HEK293
Cisapride
/dofetilide
37
-80
500 ms, 40
2ms, -100
n/a, -40 to 40
Fox-2009
Canine
myocytes
37
-80
1.5s, 50
2.5 ms, -100
1.5s, -40 to 60
source
cells
Drug
temperature holding (mV)
Abbott-2009
HEK 293
A-935142
37
Abbott-2007
HEK 293
chloramine-T
January-2005
HEK 293
Belardinelli-2008
herg
15
Pulse sequences: Channel recovery from inactivation
source
cells
Drug
temperature
holding (mV)
1st pulse
(time, mV)
2nd pulse
(time, mV)
Gintant-2009
HEK 293
A-935142
37
-80
1s, 40
n/a, -120 to 40
January-2005
HEK 293
miconazole
23
-80
200 ms, 60
300ms, -100 to -20
(10 mV inc.)
Belardinelli-2008
HEK 293
ranolazine
23
-80
200 ms, 60
300 ms, -100 to -30
(10 mV inc.)
herg
16
L-type Ca++ channel assay
Ca channel
17
Intracellular buffer composition of L-type Ca++ channel study
Charge carrier
/K blocker
ID amplifier
cells
source
CsCl
resistance
internal Ca
rundown
CsCs-MS TEA-Cl MgCl2 CaCl2 CP-E tris-P
ASP
CP
ATP
chelating
GTP HEPES EGTA NaCl glucose EDTA pH
pH
buffer
0.3
Conventional
48 AxoPatch
gMyocyte Aventis
130
20
85 HEKA EPC-9
HEK293
Merck
135
89,
AxoPatch
42
HL-1
Merck
125
20
93 Multiclamp
rMyocyte Abbott
110
30
108 AxoPatch
CHO
France
lab
140
1
50
14
4
1
5
3.6
1
5
0.2
5
2
3
0.6
10
10
7.2
CsOH
10
10
7.2
n/a
10
10
7.4
n/a
10
10
7.2
CsOH
10
10
7.2
KOH
10
10
7.2
CsOH
10
5
HT (amphotericin B for perforated whole cell patch clamp)
1 Qpatch*
CHO
Chantest
130
5
1 PatchXpress CHO
Chantest
130
5
49 PatchXpress HEK293
Chantest
88 PatchXpress HEK293
Merck
130
80
20
1
20
4
1
4
50
3**
2
4
0.1
10
10
7.2
CsOH
14
4
0.3
10
10
7.2
MS
20
5
1
10
10
7.2
CsOH
5
3
7.3
KOH
K-gluc KCl
137 IonWorks
CHO
AZ
100
40
3.2
Cs-asp: Cs-aspartate, Cs-ME: Cs-methanesulphonate. TEA-Cl: tetraethylammonium chloride, CP-E (unit/ml): Creatine phosphokinase, TrisP: tris-phosphocreatine, CP: creatine phosphate, MS: methanesulfonic acid. *CdCl2 (200 UM) added at the end of exp to block Ca current
and leak calculation/**to test Ca standard, add just before recording
with 0.2 mM cAMP and 3 mM CaCl2
Ca channel
Extracellular buffer
ID Amplifier
cells
NaCl
Choline-Cl TEA-Cl KCl CsCl CaCl2
48 AxoPatch
gMyocyte
137
85 HEKA EPC-9
HEK293
88 Multiclamp
HEK293
140
89 AxoPatch
HL-1
157
BaCl2 MgCl2
HEPES
glucose TEA-OH
pH
pH
buffer
Conventional
5.4
1.8
1
10
1
10
10
0.5
10
5
0.5
10
1.8
1
10
1.8
1
150
15
10
7.4
5
7.2
n/a
7.4
TEAOH
7.4
n/a
10
7.4
NaOH
10
10
7.4
NaOH
1.2
10
11.1
7.4
NaOH
1
10
10
7.3
KOH
10
HT
1
CHO
137
49 PatxhXpress
gMyocytes
137
88 PatchXpress
HEK293
102
4
137 IonWorks
CHO
135
4
•
•
•
Qpatch
4
5.4
30
1
Ingredients of extracellular buffer solution are quite similar between Ca channel and hERG
assay except CsCl, TEA-Cl, and Choline Cl.
TEA (Tetraethylammonium): block K channel, formation and maintenance of giga seals
BaCl2= external charge carrier (to increase the amplitude of Ca channel), block other
channels like Kir2.3 and Na during recording and maximize the L-Ca.
Ca channel
Voltage protocols for Ca++ channel
ID49
ID93
Vh
(mV)
Test
(mV)
Test
(ms)
Vh
(mV)
Prepulse
(mV)
Prepulse
(ms)
Test
(mV)
Test
(ms)
-40
0
200
-80
-40
100
0
500
inactivate the Na+ and T-type channels
used to reveal state-dependent block augmentation in Cav1.2.
Ca channel
Pulse sequences: Potency
Depolarizing
(0 to 20mV, 100 to 500 ms)
Vh=-90 to -40 mV
ID
Source
Yr
Cell line
Amplifier
Temp
(C)
Vh (mV)
Prepulse
(mV)
prepulse
(ms)
Test
(mV)
Test
(ms)
Interval (s)
1, 27
Chantest
2013
CHO
QPatch
RT
-40
0
150
5
1
Chantest
2013
CHO
PatchXpress
RT
-80
10
500
n/a
42
Pfizer
2004
Myocyte
n/a
RT
-70
-40
200
10
250
47*
Chantest
2010
CHO
PatchXpress
RT
-80
10
500
10
200
49
Chantest
2010
Myocyte
PatchXpress
RT
-40
0
300
20
50
Chantest
2008
Myocyte
n/a
RT
-40
0
300
20
48
Aventis
2004
Myocyte
Axopatch
RT
-40
0
200
85
Merck
2010
HEK293
HEKA EPC-9
RT
-90
-40
10
100
88
Merck
2009
HEK293
PatchXpress
22-26
-60
20
100
88
Merck
2009
HEK293
Multiclamp
20-25
-70
10
200
89
Merck
2004
HL-1
Axoparch
23
-50
10
100
93
Abbott
2011
myocyte
MultiClamp
RT
-80
0
500
125
Acac-China
2012
rmyocyte
AxoPatch
RT
-40
0
200
137
AZ
2012
CHO
IonWorks
RT
-65
0
500
* Dr. January: no relevant articles
Ca channel
-40
100
5
Pulse sequences: Voltage dependent
ID
Protocol
Vh (mV)
prepulse
Activation
(mV)
88
Voltage dependent
-60
-60 to 90
89
Voltage dependent
-50
-40 to 50
88
Prepulse dependent
inactivation
-60
2s, -100 to 60
Activation (ms)
Test
(mV)
Test
(ms)
Interval (s)
20
100
20
• Kinetic study of L-type Ca channel
- limited to voltage dependent/ state-dependent/use-dependent protocols
• Kinetic study in HT assay- limited.
Ca channel
15
Na+ channel
Na channel
23
Intracellular buffer of Na+ channel assay
Charge carrier
/K+ blocker
ID
amplifier
cells
source
year Cs-asp CsCl
Charge carrier
/Ca++ blocker
CsF
NaF
TEA-Cl
MgCl2
KKCl
gluconate
CP
NaCl
ATP EGTA GTP HEPES pH
pH
buffer
Conventional
48
AxoPatch
gMyocytes Aventis
2004
130
5
90
AxoPatch
HEK293
January
2009
20
92
AxoPatch*
HEK293
Merck
2010
87
101 conventional CHO
AZ
2008
120 AxoPatch
tsa201
Schwartz
2012
20
122 AxoPatch
CHO
130
123 EPC7
HEK293
Patel (UV) 2004
Wang
2002
(Harvard)
30
2
120
5
20
4
20
1
110
130
2
0.5
7.2 CsOH
5
7.4 CsOH
10
7.4 CsOH
10
5
5
10
7.2
2
10
7.4 CsOH
5
7.4 CsOH
10
10
7.2 CsOH
10
5
7.2 CsOH
10
7.2 CsOH
10
7.2
5/15
100
10
5
10
1
0.1
30
KOH
HT
1
Qpatch
CHO
Chantest
2013
1
PatchXpress CHO
Chantest
2013
130
5
4
5
47
PatchXpress CHO
Chantest
2010
130
5
4
5
49
PatxhXpress gMyocytes Chantest
2010
130
5
4
5
92
PatchXpress HEK293
Merck
2010
AZ
2008
GSK
2011
101 Ionwork
CHO
121 PatchXpress* HEK293
30
120
120
5
2
5
2
3.2
20
110
100
40
10
Cs-asp: Cs-aspartate, TEA-Cl: tetraethylammonium chloride, CP: creatine phosphate
Fluoride: introduction of fluoride anions into the cell produced an irreversible block of calcium current.
Na channel
0.1
10
5
KOH
Aspartic
7.2
acid
7.3 CsOH
3
5
7.3
KOH
10
10
7.4
HCl
0.1
10
Extracellular buffer
ID
amplifier
cells
source
NaCl
NMDG Chol-Cl TEA_Cl L-AA
KCl
CsCl CaCl2 MgCl2
KH2PO4
HEPES EGTA glucose
Na2HPO4
pH
pH
buffer
Conventional
48
AxoPatch
gMyocytes Rampe
40
90
AxoPatch
HEK293
January
140
92
AxoPatch*
HEK293
Salata
10
101 conventional
CHO
Valentine
147
120 AxoPatch
tsa201
Schwartz
122 AxoPatch
CHO
123 EPC7
97
5.4
1.8
1
5
4
1.8
0.75
5
1
1.2
20
11
7.4 TEAOH
4
1.8
1
10
10
7.4
145
4
1.8
1
10
10
7.35 NaOH
Patel (UV)
130
4
1
5
5
5
7.4
HEK293
Wang
(Harvard)
65
Kramer
137
4
1.8
1
10
10
7.4
4
1.8
1
10
10
7.4 NMDG
4
1.8
1
10
10
7.4 NMDG
1
2.7
0.5
5
2.7/140
0.9
0.5/1
4
2
1
125
5
85
2
10
7.4 NMDG
7.4
10
NaOH
NaOH
NaOH
7.4 TEAOH
HT
1
Qpatch/PX
CHO
49
PatxhXpress
gMyocytes Brown
40
49
PatxhXpress
gMyocytes Brown
40
92
PatchXpress
HEK293
Salata
40
101 Ionwork*
CHO
Valentine
138
121 PatchXpress*
HEK293
GSK
35
•
•
97
120
105
1.5/8.1
20
10
1
10
NaOH
7.5
HCl
7.3
KOH
7.35
HCl
NMDG (N-methyl-D-glucamine), lower mobility, produce series resistances larger (by about 30-50%) than Cs.
L-AA: L-aspartic acid
Na channel
Pulse sequences: Potency
Depolarizing
(-30 to 0 mV, 10-200 ms)
Hyperpolarizing
(-120 mV)
holding
(-150 to -80 mV)
ID
Source
Yr
Vh
(mV)
Hyperpolarizing
(pre-pulse)
(mV)
Depolarizing
(Test)
( ms)
(mV)
(ms)
-10
200
Vh II
(mV)
Depolarizing II
(Test)
( ms)
(mV)
Interval (s)
(ms)
1
Chantest
2013
-80
-120
47
Chantest*
2010
-80
-120
500
-10
200
49
Chantest
2010
-80
-120
20
-15
10
48
Aventis
2004
-110
-20
n/a
10
50
Chantest
2008
-80
-15
10
10
92
MerCk
2010
-100
-20
30
5
95
January
2009
-140
-20
24
10
96
January
2008
-150
-20
24
15
101
AZ
2008
-120
0
n/a
120
Schwartz
2012
-120
-10
n/a
121
GSK
2011
-30
50
122
UV
2004
-120
20
25
125
Zhang
2012
-100
-20
30
-120
-120
* Two step pulse for tonic and phasic block
200
200
Na channel
10
-80
200
-10
200
1
50
Pulse sequences: Voltage dependent
ID
Source
Yr
Vh (mV)
Hyperpolarizing
(pre-pulse)
(mV)
Depolarizing
(Test)
( ms)
(mV)
(ms)
92
Salata
2010
-120
-90 to 30
30
100
Mekielski
1998
-150
-90 to 30
24
101
Valentine
2008
-120
-90 to 90
50
Na channel
Pulse sequences: channel kinetic protocols
ID
Source
Yr
kinetics
Vh
(mV)
Hyperpolarizing
or pre-pulse
(mV)
Depolarizing
(Test)
( ms)
Vh II
(mV)
(ms)
Depolarizing II
(Test)
(mV)
( ms)
(mV)
(ms)
-120
500
20
24
-20
24/240
-15
10
90
Mayo
2009
activation
-140
-120 to 60
n/a
100
UC
1998
activation
-150
various
24
121
GSK
2011
activation
-120
-90 to -35
32
122
UV
2004
activation
-120
-80 to 60
20
90
Mayo
2009
inactivation
-140
-150 to 0
1000
0
24
121
GSK
2011
inactivation
-120
-130 to -40
480
-30
50
100
UC
1998
inactivation
-120
-150 to -30
1000
122
UV
2004
Inactivation
-120
-120 to -20
1000
20
n/a
100
UC
1998
recovery
-120
-20
1000
-120
var
50
Chantest
2008
recovery
-80
-15
500
-120
var
122
UV
2004
recovery
-120 to 20
100
-90
var
20
123
Harvard
2002
recovery
-70
10
-140
var
30
-140
Na channel
n/a
500
5