1. Protocols of cardiac ion channels hERG, Ca++ and Na+
CIPA project
Protocols of cardiac ion channels hERG, Ca++ and Na+
Eunjung Park
FDA/CDER/OND/DCRP
herg

2. 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
Cells
Manual
HEK293 (12) > CHO (3) > myocyte (1) > mouse L-cell (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)
Temp
Near Physiological (8) > Room temperature (6) RT
Recording
Whole-cell patch clamp
Whole cell parch clamp
Whole cell patch clamp or perforated patch clamp
Literature search
Primarily focused on major pharmaceutical companies and Chantest which have publications on cardiac ion channels. Therefore, this summary is not necessarily represent whole channel assay methods.
Total 70 manual ion channel assay protocols were reviewed. When multiple literatures with same protocols from same groups or authors were found, a representative protocol was included for a summary. In case of the same groups used different protocols, all different protocols were accepted for this analysis.
Some of the literatures have multiple ion channel assay using different cells. Therefore, the sum of numbers of sources of channel (cells) are not equal to the number of cited literatures.
hERG assay
Sources of channel: minor cell lines are mouse L-cell (fibroblast) and SH-SY5Y (human neuroblastoma cell line).
Majority of manual and PatchXpress assay used HEK293 but HT assays using IonWorks system used CHO.
Assay temperature is 60 vs 40 for near physiological temperature and room temperature.
IonWork system used perforated whole cell patch clamp.
Ca channel assay
Manual cardiac Ca channel assay from selected literatures used myocytes more than transfected cell lines.
All assays were done at room temperature.
Na channel assay
Major cell lines for manual assay is HEK293.
summary

3. 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 *
Assay condition of manual patch clamp in terms of pipet resistance and seal resistance.
Total 70 literatures were collected for hERG, Ca and Na channel protocols. 96% of the protocols indicated pipet tip resistance, 4.3% of protocols for seal resistance and 50% of protocols showed series resistance compensation.
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

4. Seal resistance in automatic patch clamp assays
PatchXpress
IonWork
Barracuda
IonWork HT
Ref ID (yr)
88 (2009)
92 (2010)
121 (2011)
151 (2005)
61 (2013)
62 (2006)
101 (2008)
Ion channel
Ca++
Na+
hERG/Na+
hERG
Sponsor
Merck
GSK
J&J AZ
Cell
HEK293
CHO
Re (M)
1-3
1-9
(ave 2.4)
1-2.8
(ave 1.7)
Rseal (M)
2520
>1000
1083
1900
> 30
(ave. 58)
> 60
> 60 (HT)
> 30 (Quattro)
Rm (M)
1358
>150
>200
(ave 710)
Ra (M)
11.8
<15
(ave. <10) 14
Membrane rupture
suction
amphotericin
Amphotericin
Quattro (Escin)
Seal resistance in automatic assay are different with manual.
Major automatic assay from selected literatures used PatchXpress and Ionworks.
PatchXpress: whole cell patch clamp, gigaohm seals, 16 wells in parallel, 1cell/hole per well
IonWorks: perforated whole cell patch clamp, ~100 M seal on IonWorks HT and M seal on Quattro, 384 well: 48 channels sequentially, 1(HT) or 64 (Quattro) cells/ holes per well
Rseal: Patch seal resistance, Rm: membrane resistance during whole cell recording, Ra: whole cell access resistance, Re: electrode resistance
summary

5. Extracellular buffer
hERG
hCav1.2
hNav1.5 M A
NaCl 
KCl
CaCl2*
MgCl2
HEPES
glucose
KH2PO4
()
Na2HPO4
NaHCO3
EGTA
NMDG
CsCl
Cholin-Cl
TEA-Cl
BaCl2*
L-aspartic acid
Ingredients of extracellular buffer are similar across the hERG, Ca and Na but some other special ingredients were added to improve the assay quality.
NMDG: stronger seal resistance
TEA-Cl: stronger seal resistance
BaCl2
substitute Ca because Ba is better to maintain background and leak current small.
BaCl2 block Na channel
M: conventional manual patch clamp (mainly, Axopatch)
A: Automatic patch clamp
(): included in less than half of protocols
summary

6. Intracellular buffer
hERG
hCav1.2
hNav1.5 M A
Current carrier
K-aspartate
K-gluconate* 
() Cs
(K block)
CsCl
Cs-aspartate
Cs-methanesulphonate
Fluoride
(Ca block) KF
NaF
CsF
KCl*
MgCl2
EGTA
HEPES
GTP
ATP
Na2ATP
K2ATP
MgATP
regeneration system
Creatine phosphokinase
Creatine phosphate
Tris-phosphoreatine
Seal resistance
TEA (tetraehtylammonium)
CaCl2
NaCl**
Cs: current carrying and K block (not completely, remnant K current can be blocked by Cd, intrendipine and conotoxon)
KF improve the success rate of PatchXpress-hERG assay.
TEA, Tris, NMDG (N-methyl-D-glucamine): low mobility but produce seal resistance larger
Ca: low Ca level makes Ca current longer
EGTA: chelating trace amount of heavy metal and improve ca current
ATP and cAMP : reverse rundown
ATP regenerating system: slow rundown
M: conventional manual patch clamp (mainly, Axopatch) A: Automatic patch clamp, (): included in less than half of protocols
summary

7. Representative pulse sequences for a drug evaluation
Depolarizing
(0 to 70 mV, 1-4s)
Repolarizing
(-80 to 20 mV, 2-6s)
hERG
Vh=-80 mV
Vh=-90 to -40 mV
Depolarizing
(0 to 20mV, 100 to 500 ms)
hCav1.2
Common pulse sequence to measure IC50 of drugs.
hERG: holding-depolarization-repolarization (tail current measure)
Ca: holding-depolarization (measure)-holding
Na: holding-hyperpolarizing-depolarization (measure)
Major Vh (holding potential): hERG, -80mV, Ca, -40mV, Na, -80mV
Depolarizing: hERG, 20 or 40 mV, Ca, 0 to 20 mV, Na, -30 to 0 mV
Sodium channel is needed hyperpolarizing step.
Depolarizing
(-30 to 0 mV, ms)
Hyperpolarizing
(-120 mV)
hNav1.5
Vh=-150 to -80 mV
Ref: JCE 2010, 21, 301
summary

8. hERG assay
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9. Representative intracellular buffer solution
 Source
Year
Amplifier
Intracellular (pipet) (mM)
K-aspartate
K-gluconate
KCl
EGTA
ATP KF
HEPES
MgCl2
CaCl2
NaCl pH
pH buffer
manual
Chantest
2013
AxoPatch
130 5 4 10
7.2
KOH
Merck
2006
119 15
3.2
7.35
Abbott
2012
125 20 1
7.3
UWM
Automatic
GSK
Barracuda
140
PatchXpress
0.1
QPatch
100
2008 60 70 AZ
IonWorks 40 3
hERG buffer solutions are pretty much consistent.
Extracellular buffer solution (cell superfusion) mimics extracellular components. Main component is NaCl and milimolar Ca and Mg are necessary. pH
Intracellular buffer solution (pipette solution) mimics cytosol. K+, organic anion, and Cl- are major component. ATP and/or GTP is added to maintain intact cellular function. pH 7.2
KF is (1) similar to those with standard potassium chloride solutions, (2) use of KF significantly improves the success rate of hERG screening using PatchXpress without compromising data quality, and (3) utilization of KF can significantly increase the throughput of hERG screening with PatchXpress.
UWM: University of Wisconsin at Madison
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10. Representative extracellular buffer solution
 Source
Year
amplifier
Extracellular (superfusion) (mM)
NaCl
KCl
CaCl2
MgCl2
HEPES
glucose
NaHCO3 pH
pH buffer
Manual
Chantest
2013
AxoPatch
137 4
1.8 1 10
7.4
NaOH
Merck
2006
132
1.2
11.1
7.35
Abbott
2012
140 5 2 20
UWM
Automatic
GSK
Barracuda
136 3 6 12
PatchXpress
QPatch
145
2008
0.5 11 AZ
IonWorks
7.3
hERG buffer solutions are pretty much consistent.
Extracellular buffer solution (cell superfusion) mimics extracellular components. Main component is NaCl and milimolar Ca and Mg are necessary. pH
Intracellular buffer solution (pipette solution) mimics cytosol. K+, organic anion, and Cl- are major component. ATP and/or GTP is added to maintain intact cellular function. pH 7.2
UWM: University of Wisconsin at Madison
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11. Pulse sequences: potency
Source-Yr
temperature
sample replication
pulse
holding (mV)
1st pulse
(time, mV)
2nd pulse
interval
Chantest-2013
ambient
n/a
step
-80
2s, 40
2s, -40
10s
NDA- 2007
35±2
>3
step-ramp
1s, 20
0.5mV/ms, +20 to -80 5s
Merck-2006
35 ± 0.5
5-8
2s, -50
15s
NDA- 2004 35
Abbott-2012* 5
Step-ramp
1.5s, 0
1mV/25ms, 0 to -80
NDA- 2002 6
3s, 0
4s, -50
Dr. January-2009
23 ± 1
3-6
4s, 70
5.7s, -50
Pfizer/Univ Walk-2010 37
2s, 20
4s, -40
12s
0.5mV/ms, to -80
Typical pulse sequences of pharmacology study. Vh is consistently -80mV.
The pulse sequence in the NDA submission of each source was extracted to compare.
Chantest: most recent literature (2013, Scientific reports) used room temperature and single step sequence but hERG assay(2007) for NDA submission was done at near physiological temperature and step-ramp protocols.
Merck: (hERG assay for NDA submission) and 2006 data (literature) showed same pulse sequence.
Abbott: 2012 literature used step-ramp sequence and 2002 assay for NDA submission used step protocol.
Dr. January from University of Wisconsin used almost same protocols repeatedly.
* hERG enhancer
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12. Pulse sequences: Voltage dependent
source
cells
Drug
temperature
holding (mV)
1st pulse
(time, mV)
2nd pulse
Abbott-2009
HEK 293 37
-80
n/a, -60 to 40
n/a. -100
Abbott-2007
HEK293
chloramine-T
3s, -60 to 40
(10mV inc)
4s, -60
January-1998 35
4s, -60 to 50
5s, -50
January-2005 23
4s, -70 to 60
(10 mV/15s)
6s, -50
Hancox-2010
Dofetilide/cisapride
2s, -40 to 50
4s, -40
Belardinelli-2008
ranolazine
4s, -80 to 70
( 10mV inc.)
5.7s, -50
Fox-2009
Canine myocytes
0.5s, 60 to -30
(10 mV inc)
1.6s, -30
Voltage dependent protocols
Typical voltages dependent protocol used Vh (-80), activation using the range of -80 to 70 mV and deactivation.
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13. Pulse sequences: Channel activation
source
Drug
temperature
holding (mV)
1st pulse
(time, mV)
2nd pulse
Abbott-2009
A * 37
-80
5-850 ms (30ms inc), -10
n/a, -100
Abbott-2007
chloramine-T
5-185 ms, 30
5-480 ms, 0
January-2005
Miconazole 23
various duration, 20
1s, -50
* hERG enhancer
Activation protocols.
Various duration of depolarization at fixed voltage.
Overall, duration of pulse is shorter than voltage dependent protocols.
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14. Pulse sequences: Channel deactivation
source
Drug
temperature
holding (mV)
1st pulse
(time, mV)
2nd pulse
Abbott-2009
A * 37
-80
n/a, 40
n/a, -70 to -120
Merck-2006
Flunarizine 35
1s, 50
2s, -120 to 20
(10mV inc)
January-2005
miconazole 23
1s, 60
5s, to -20
(10 mV inc)
* hERG enhancer
Deactivation protocols.
Single fixed activation and ramp in repolarization pulse used.
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15. Pulse sequences: Channel inactivation
source
cells
Drug
temperature
holding (mV)
1st pulse
(time, mV)
2nd pulse
3rd pulse
Abbott-2009
HEK 293 A 37
-80
500ms, 60
2 ms, -100
n/a, -40 to 60
Abbott-2007
chloramine-T
n/a, -20 to 60
January-2005
miconazole 23
200 ms, 60
100ms, -100
300ms, -20 to 60
Belardinelli-2008
ranolazine
300 ms, 60
10ms, -100
n/a, -40 to 40
(10 mV inc.)
Pfizer/Univ-2010
HEK293
Cisapride
/dofetilide
500 ms, 40
2ms, -100
Fox-2009
Canine myocytes
1.5s, 50
2.5 ms, -100
1.5s, -40 to 60
Single fixed activation and short hyperpolarization then, various range of inactivation pulse.
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16. Pulse sequences: Channel recovery from inactivation
source
cells
Drug
temperature
holding (mV)
1st pulse
(time, mV)
2nd pulse
Gintant-2009
HEK 293 A 37
-80
1s, 40
n/a, -120 to 40
January-2005
miconazole 23
200 ms, 60
300ms, to -20
(10 mV inc.)
Belardinelli-2008
ranolazine
300 ms, -100 to -30
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17. L-type Ca++ channel assay
T-type ca channel was not included in this summary.
Ca channel

18. Intracellular buffer composition of L-type Ca++ channel study
Charge carrier
/K blocker
resistance
internal Ca
rundown
chelating ID
amplifier
cells
source
CsCl
Cs-ASP
Cs-MS
TEA-Cl
MgCl2
CaCl2
CP-E
tris-P CP
ATP
GTP
HEPES
EGTA
NaCl
glucose
EDTA pH
pH buffer
Conventional 48
AxoPatch
gMyocyte
Aventis
130 20 1 50 14 4
0.3 10
7.2
CsOH 85
HEKA EPC-9
HEK293
Merck
135 5
n/a
89, 42
HL-1
125
3.6
0.2
7.4 93
Multiclamp
rMyocyte
Abbott
110 30
108
CHO
France lab
140 2 3
0.6
KOH
HT (amphotericin B for perforated whole cell patch clamp)
Qpatch*
Chantest
PatchXpress
0.1 49 MS 88 80
3**
K-gluc
KCl
137
IonWorks AZ
100 40
3.2
7.3
The common ingredients of intracellular buffer composition between Ca and hERG= MgCl2, HEPES, ATP
Main difference = GTP, Cs instead of K
Fast rundown of Ca++ channel is a difficulty to develop the assay method.
To slow down the rundown, ATP or ATP regenerating system and GTP were used.
TEA-Cl: maintain high seal resistance.
The literature from AZ 2012 (Am J Physiol Heart Circ Physiol 302, H1466) indicated that they used K-gluconate and KCl for Na+ and Ca++ channel assay.
Cs-asp: Cs-aspartate, Cs-ME: Cs-methanesulphonate. TEA-Cl: tetraethylammonium chloride, CP-E (unit/ml): Creatine phosphokinase, Tris-P: 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

19. Extracellular buffer ID
Amplifier
cells
NaCl
Choline-Cl
TEA-Cl
KCl
CsCl
CaCl2
BaCl2
MgCl2
HEPES
glucose
TEA-OH pH
pH buffer
Conventional 48
AxoPatch
gMyocyte
137
5.4
1.8 1 10
7.4 85
HEKA EPC-9
HEK293
150 15 5
7.2
n/a 88
Multiclamp
140
0.5
TEAOH 89
HL-1
157 HT
Qpatch
CHO 4
NaOH 49
PatxhXpress
gMyocytes
PatchXpress
102 30
1.2
11.1
IonWorks
135
7.3
KOH
Common ingredients with hERG = NaCl, KCl, CaCl2, MgCls, HEPES, glucose.
In some manual assay, Choline-Cl or TEA-Cl was used to substitute for NaCl.
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

20. Voltage protocols for Ca++ channel
ID49
ID93
Vh (mV)
Test
(mV)
Test (ms)
-40
200
Vh (mV)
Prepulse
(mV)
Prepulse (ms)
Test
Test (ms)
-80
-40
100
500
Typical pulse sequence to evaluate drug activity on Ca channel is one step activation (depolarization).
Vh is usually -40 mV where Na and T-type Ca channels are inactivated at.
If Vh is -80 mV, pre-pulse at -40mV was set to block other currents.
inactivate the Na+ and T-type channels
used to reveal state-dependent block augmentation in Cav1.2.
Ca channel

21. Pulse sequences: Potency
Vh=-90 to -40 mV
Depolarizing
(0 to 20mV, 100 to 500 ms) 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
150 5 1
PatchXpress
-80 10
500
n/a 42
Pfizer
2004
Myocyte
-70
200
250
47*
2010 49
300 20 50
2008 48
Aventis
Axopatch 85
Merck
HEK293
HEKA EPC-9
-90
100 88
2009
22-26
-60
Multiclamp
20-25 89
HL-1
Axoparch 23
-50 93
Abbott
2011
myocyte
MultiClamp
125
Acac-China
2012
rmyocyte
AxoPatch
137 AZ
IonWorks
-65
Generally Vh is -40 and depolarization was set at 0 mV.
The pulse sequence of automatic system is not necessarily different with manual assay system.
Ca channel
* Dr. January: no relevant articles

22. Pulse sequences: Voltage dependentID
Protocol
Vh (mV)
prepulse
Activation
(mV)
Activation (ms)
Test
Test (ms)
Interval (s) 88
Voltage dependent
-60
-60 to 90 20 15 89
-50
-40 to 50
100
Prepulse dependent inactivation
2s, to 60
Kinetic study of L-type Ca channel
- limited to voltage dependent/ state-dependent/use-dependent protocols
Kinetic study in HT assay- limited.
The kinetic protocols of L-type Ca channel was not found from selected literatures.
There are voltage-dependent, state-dependent and use-dependent protocols available.
Kinetic study in the high throughput assay system is somewhat limited. For example, it is hard to make customized protocol for one drug such as specific treatment time.
Ca channel

23. Na+ channel
Late Na channel protocols were not included in this summary.
Na channel

24. Intracellular buffer of Na+ channel assay
Charge carrier
/K+ blocker
Charge carrier
/Ca++ blocker ID
amplifier
cells
source
year
Cs-asp
CsCl
CsF
NaF
TEA-Cl
MgCl2
K-gluconate
KCl CP
NaCl
ATP
EGTA
GTP
HEPES pH
pH buffer
Conventional 48
AxoPatch
gMyocytes
Aventis
2004
130 5 2
0.1 10
7.2
CsOH 90
HEK293
January
2009 20
120
7.4 92
AxoPatch*
Merck
2010 87 4
0.5
101
conventional
CHO AZ
2008 1
KOH
tsa201
Schwartz
2012
110
122
Patel (UV)
5/15
123
EPC7
Wang (Harvard)
2002
100 30 HT
Qpatch
Chantest
2013
PatchXpress 47 49
PatxhXpress
Aspartic acid
7.3
Ionwork
3.2 40 3
121
PatchXpress*
GSK
2011
HCl
The presence of fluoride in the intracellular solution promotes development of high resistance seals as well as seal stability. As with conventional patch clamp, the
osmolality of solutions (typically mOsmol) is also important for seal quality and longevity on the PatchXpress.
In addition, fluoride blocks Ca++ channel.
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

25. Extracellular buffer ID
amplifier
cells
source
NaCl
NMDG
Chol-Cl
TEA_Cl
L-AA
KCl
CsCl
CaCl2
MgCl2
KH2PO4
Na2HPO4
HEPES
EGTA
glucose pH
pH buffer
Conventional 48
AxoPatch
gMyocytes
Rampe 40 97
5.4
1.8 1 5 10
7.4 90
HEK293
January
140 4
0.75
NaOH 92
AxoPatch*
Salata
125
1.2 20 11
TEAOH
101
conventional
CHO
Valentine
147
120
tsa201
Schwartz
145
7.35
122
Patel (UV)
130
123
EPC7
Wang (Harvard) 65 85 2 HT
Qpatch/PX
Kramer
137 49
PatxhXpress
Brown
PatchXpress
2.7
0.5
7.5
HCl
Ionwork*
138
2.7/140
0.9
0.5/1
1.5/8.1
7.3
KOH
121
PatchXpress*
GSK 35
105
Basic extracellular buffer components are similar across the literarures.
NMDG (N-methyl-D-glucamine), lower mobility, produce series resistances larger (by about 30-50%) than Cs.
L-AA: L-aspartic acid
Na channel

26. Pulse sequences: Potency
Hyperpolarizing
(-120 mV)
holding
(-150 to -80 mV)
Depolarizing
(-30 to 0 mV, ms) ID
Source Yr
Vh (mV)
Hyperpolarizing
(pre-pulse)
Depolarizing
(Test)
Vh II
Depolarizing II
Interval (s)
(mV)
( ms)
(ms) 1
Chantest
2013
-80
-120
-10
200 10 47
Chantest*
2010
500 49 20
-15 48
Aventis
2004
-110
-20
n/a 50
2008 92
MerCk
-100 30 5 95
January
2009
-140 24 96
-150 15
101 AZ
120
Schwartz
2012
121
GSK
2011
-30
122 UV 25
125
Zhang
Basically Vh is -80 and pre-pulse for hyperpolarization is necessary.
Depolarizing step was set between -30 to 0 mV.
* Two step pulse for tonic and phasic block
Na channel

27. Pulse sequences: Voltage dependentID
Source Yr
Vh (mV)
Hyperpolarizing
(pre-pulse)
Depolarizing
(Test)
(mV)
( ms)
(ms) 92
Salata
2010
-120
-90 to 30 30
100
Mekielski
1998
-150 24
101
Valentine
2008
-90 to 90 50
Instead of pre-pulse, Vh is set at -120 mV. Depolarization voltage step is ranging from -90 to 90 mV.
Na channel

28. Pulse sequences: channel kinetic protocolsID
Source Yr
kinetics
Vh (mV)
Hyperpolarizing
or pre-pulse
Depolarizing
(Test)
Vh II
Depolarizing II
(mV)
( ms)
(ms) 90
Mayo
2009
activation
-140
-120 to 60
n/a
100 UC
1998
-150
various 24
121
GSK
2011
-120
-90 to -35 32
122 UV
2004
-80 to 60 20
inactivation
-150 to 0
1000
-130 to -40
480
-30 50
-150 to -30
500
Inactivation
-120 to -20
recovery
-20
var
24/240
Chantest
2008
-80
-15 10
-120 to 20
-90
123
Harvard
2002
-70 30 5
Ref 90, Mayo clinic, Dr. January, and Dr. Makielski
UW: University of Wisconsin, Dr. January
UC: University of Chicago, Dr. Makielski
UV: University of Virginia, Dr. Patel
Basic pulse sequence of protocols for activation, inactivation and recovery from inactivation is similar across the research teams.
Na channel