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

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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

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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

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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