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CIPA project. Protocols of cardiac ion channels hERG, Ca ++ and Na +. Eunjung Park FDA/CDER/OND/DCRP. Summary of protocols. *. *. Access and seal resistance of manual patch clamp assays. *. Pipette (tip) resistance (access resistance, electrode resistance): - PowerPoint PPT Presentation
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Protocols of cardiac ion channels hERG, Ca++ and Na+
Eunjung ParkFDA/CDER/OND/DCRP
herg 1
CIPA projectCIPA project
Summary of protocols
summary 2
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
CellsManual
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)
TempManual Near Physiological (8) >
Room temperature (6) RT RT
Automatic RT RT RT
Recording 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
Access and seal resistance of manual patch clamp assays
**
*
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%).
Pipette (tip) resistance (access resistance, electrode resistance):• < 5M in 67 out of 70 literatures (96%) including hERG, Ca++ and Na+ channels
summary 3
Seal resistance in automatic patch clamp assays
PatchXpress IonWorkBarracuda 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
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 >200(ave 710)
Ra (M) 11.8 <15 (ave. <10)
<15 14
Membrane rupture suction suction suction suction amphotericin amphotericin Amphotericin
Quattro (Escin)
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 hCav1.2 hNav1.5
M A M A M A
Current carrier K-aspartateK-gluconate*
()
Cs (K block)
CsClCs-aspartateCs-methanesulphonate
Fluoride(Ca block)
KFNaFCsF
()
KCl* () ()
MgCl2
EGTA
HEPES
GTP ()
ATP Na2ATPK2ATPMgATP
()
ATPregeneration system
Creatine phosphokinaseCreatine phosphateTris-phosphoreatine
() () ()
Seal resistance TEA (tetraehtylammonium) ()
CaCl2 () ()
NaCl** () () ()
summary 6
M: conventional manual patch clamp (mainly, Axopatch) A: Automatic patch clamp, (): included in less than half of protocols
Representative pulse sequences for a drug evaluation
hERG
hCav1.2
hNav1.5
Vh=-80 mV
Vh=-150 to -80 mV
Hyperpolarizing (-120 mV)
Depolarizing(0 to 70 mV, 1-4s)
Depolarizing(-30 to 0 mV, 10-300 ms)
Repolarizing(-80 to 20 mV, 2-6s)
Vh=-90 to -40 mV
Depolarizing(0 to 20mV, 100 to 500 ms)
summary 7
Ref: JCE 2010, 21, 301
hERG assay
herg 8
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 5 7.2 KOH
Merck 2006 AxoPatch 119 15 5 5 5 3.2 7.35 KOH
Abbott 2012 AxoPatch 125 20 10 5 5 1 7.3 KOH
UWM 2006 AxoPatch 130 5 5 10 1 7.2 KOH
AutomaticGSK 2013 Barracuda 140 20 1 1 7.35 KOHGSK 2013 PatchXpress 130 10 5 5 10 1 0.1 7.2 KOHGSK 2013 QPatch 100 10 5 10 10 1 1 7.2 KOH
Merck 2008 PatchXpress 60 5 70 5 15 7.2AZ 2012 IonWorks 140 1 20 1 7.3 KOHAZ 2012 IonWorks 100 40 3 5 3.2 7.3 KOH
Representative intracellular buffer solution
UWM: University of Wisconsin at Madison
herg 9
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 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
Automatic
GSK 2013 Barracuda 136 3 2 1 20 6 12 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 NaOH
UWM: University of Wisconsin at Madison herg 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 Walk-2010 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
source cells Drug temperature holding (mV)
1st pulse (time, mV)
2nd pulse (time, mV)
Abbott-2009 HEK 293 37 -80 n/a, -60 to 40 n/a. -100
Abbott-2007 HEK293 chloramine-T 37 -80 3s, -60 to 40 (10mV inc) 4s, -60
January-1998 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
herg 12
Pulse sequences: Voltage dependent
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
source cells Drug temperature holding (mV) 1st pulse (time, mV)
2nd pulse (time, mV)
3rd pulse (time, mV)
Abbott-2009 HEK 293 A-935142 37 -80 500ms, 60 2 ms, -100 n/a, -40 to 60
Abbott-2007 HEK 293 chloramine-T 37 -80 500ms, 60 2 ms, -100 n/a, -20 to 60
January-2005 HEK 293 miconazole 23 -80 200 ms, 60 100ms, -100 300ms, -20 to 60
Belardinelli-2008 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
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
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 10 7.2 CsOH
85 HEKA EPC-9 HEK293 Merck 135 1 5 10 10 7.2 n/a
89, 42 AxoPatch HL-1 Merck 125 20 3.6 5 0.2 10 10 7.4 n/a
93 Multiclamp rMyocyte Abbott 110 30 1 5 10 10 10 5 7.2 CsOH
108 AxoPatch CHO France lab 140 2 3 0.6 10 10 7.2 KOH
HT (amphotericin B for perforated whole cell patch clamp)
1 Qpatch* CHO Chantest 130 5 1 4 10 10 2 7.2 CsOH
1 PatchXpress CHO Chantest 130 5 4 0.1 10 10 7.2 CsOH
49 PatchXpress HEK293 Chantest 130 20 1 50 14 4 0.3 10 10 7.2 MS
88 PatchXpress HEK293 Merck 80 20 4 3** 20 5 1 10 10 7.2 CsOH
K-gluc KCl
137 IonWorks CHO AZ 100 40 3.2 5 3 7.3 KOH
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
Charge carrier/K blocker rundownresistance internal Ca chelating
Ca channel
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 10 7.4
85 HEKA EPC-9 HEK293 150 15 1 10 5 7.2 n/a
88 Multiclamp HEK293 140 10 0.5 10 10 7.4 TEAOH
89 AxoPatch HL-1 157 5 0.5 10 7.4 n/a
HT
1 Qpatch CHO 137 4 1.8 1 10 10 7.4 NaOH
49 PatxhXpress gMyocytes 137 5.4 1.8 1 10 10 7.4 NaOH
88 PatchXpress HEK293 102 4 30 1.2 10 11.1 7.4 NaOH
137 IonWorks CHO 135 4 1 10 10 1 7.3 KOH
• 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++ channelID49 ID93
Vh (mV)
Test(mV)
Test (ms)
-40 0 200
Vh (mV)
Prepulse(mV)
Prepulse (ms)
Test(mV)
Test (ms)
-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
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 5
89 Merck 2004 HL-1 Axoparch 23 -50 10 100
93 Abbott 2011 myocyte MultiClamp RT -80 -40 100 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
Vh=-90 to -40 -40 mV
Depolarizing(00 to 20mV, 100 to 500 ms)
Ca channel
Pulse sequences: Voltage dependent
ID Protocol Vh (mV) prepulse Activation(mV)
Activation (ms) Test(mV)
Test (ms)
Interval (s)
88 Voltage dependent -60 -60 to 90 20 15
89 Voltage dependent -50 -40 to 50 100
88 Prepulse dependent inactivation -60 2s, -100 to 60 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
Na+ channel
Na channel 23
Intracellular buffer of Na+ channel assay
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 AxoPatch HEK293 January 2009 20 120 5 2 5 7.4 CsOH
92 AxoPatch* HEK293 Merck 2010 87 20 4 20 5 10 0.5 10 7.4 CsOH
101 conventional CHO AZ 2008 1 130 5 5 10 7.2 KOH
120 AxoPatch tsa201 Schwartz 2012 20 110 10 2 10 7.4 CsOH
122 AxoPatch CHO Patel (UV) 2004 130 1 5/15 5 7.4 CsOH
123 EPC7 HEK293 Wang (Harvard) 2002 100 30 10 10 7.2 CsOH
HT
1 Qpatch CHO Chantest 2013 30 120 5 2 10 5 7.2 CsOH
1 PatchXpress CHO Chantest 2013 130 5 4 5 0.1 10 7.2 CsOH
47 PatchXpress CHO Chantest 2010 130 5 4 5 10 7.2 KOH
49 PatxhXpress gMyocytes Chantest 2010 130 5 4 5 0.1 10 7.2 Aspartic acid
92 PatchXpress HEK293 Merck 2010 30 120 5 2 10 5 7.3 CsOH
101 Ionwork CHO AZ 2008 3.2 100 40 3 5 7.3 KOH
121 PatchXpress* HEK293 GSK 2011 20 110 10 10 10 7.4 HCl
Cs-asp: Cs-aspartate, TEA-Cl: tetraethylammonium chloride, CP: creatine phosphateFluoride: introduction of fluoride anions into the cell produced an irreversible block of calcium current.
Charge carrier/Ca++ blocker
Charge carrier/K+ blocker
Na channel
Extracellular bufferID 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 NMDG
90 AxoPatch HEK293 January 140 4 1.8 0.75 5 7.4 NaOH
92 AxoPatch* HEK293 Salata 10 125 5 1 1.2 20 11 7.4 TEAOH
101 conventional CHO Valentine 147 4 1.8 1 10 10 7.4 NaOH
120 AxoPatch tsa201 Schwartz 145 4 1.8 1 10 10 7.35 NaOH
122 AxoPatch CHO Patel (UV) 130 4 1 5 5 5 7.4 NaOH
123 EPC7 HEK293 Wang (Harvard) 65 85 2 10 7.4 TEAOH
HT
1 Qpatch/PX CHO Kramer 137 4 1.8 1 10 10 7.4 NaOH
49 PatxhXpress gMyocytes Brown 40 4 1.8 1 10 10 7.4 NMDG
49 PatxhXpress gMyocytes Brown 40 97 4 1.8 1 10 10 7.4 NMDG
92 PatchXpress HEK293 Salata 40 120 1 2.7 0.5 5 7.5 HCl
101 Ionwork* CHO Valentine 138 2.7/140 0.9 0.5/1 1.5/8.1 20 1 7.3 KOH
121 PatchXpress* HEK293 GSK 35 105 4 2 1 10 10 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
ID Source Yr Vh (mV)
Hyperpolarizing (pre-pulse)
Depolarizing(Test)
Vh II Depolarizing II(Test)
Interval (s)
(mV) ( ms) (mV) (ms) (mV) ( ms) (mV) (ms)
1 Chantest 2013 -80 -120 -10 200 10
47 Chantest* 2010 -80 -120 500 -10 200 -80 200 -10 200 1
49 Chantest 2010 -80 -120 20 -15 10
48 Aventis 2004 -110 -20 n/a 10
50 Chantest 2008 -80 -120 200 -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 50
121 GSK 2011 -120 200 -30 50
122 UV 2004 -120 20 25
125 Zhang 2012 -100 -20 30
Hyperpolarizing (-120 mV)
holding(-150 to -80 mV)
Depolarizing(-30 to 0 mV, 10-200 ms)
* Two step pulse for tonic and phasic block Na channel
ID Source Yr Vh (mV) Hyperpolarizing (pre-pulse)
Depolarizing(Test)
(mV) ( 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: Voltage dependent
Pulse sequences: channel kinetic protocols
ID Source Yr kinetics Vh (mV)
Hyperpolarizing or pre-pulse
Depolarizing(Test)
Vh II Depolarizing II(Test)
(mV) ( ms) (mV) (ms) (mV) ( ms) (mV) (ms)
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 -120 500 20 24
122 UV 2004 Inactivation -120 -120 to -20 1000 20 n/a
100 UC 1998 recovery -120 -20 1000 -120 var -20 24/240
50 Chantest 2008 recovery -80 -15 500 -120 var n/a 500 -15 10
122 UV 2004 recovery -120 to 20 100 -90 var 20
123 Harvard 2002 recovery -140 -70 10 -140 var 30 5
Na channel