IonWorks Quattro Assay - Molecular Devices
IonWorks Quattro Assay - Molecular Devices
IonWorks Quattro Assay - Molecular Devices
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<strong>IonWorks</strong> <strong>Assay</strong>s for Nav Channels<br />
Biophysical Society<br />
MDS Automated EP User meeting<br />
Mark R. Bowlby, PhD<br />
Discovery Neuroscience<br />
January 31, 2008
Outline<br />
n Na + Channels in Disease<br />
n <strong>IonWorks</strong> HT vs. FLIPR <strong>Assay</strong>s<br />
n Pharmacology on <strong>IonWorks</strong><br />
n <strong>IonWorks</strong> <strong>Quattro</strong> Performance<br />
n Another Reality
Sodium Channel Complexes<br />
n<br />
n<br />
α1 (>200KDa, 9 subtypes)<br />
Pore-forming<br />
β (4 subtypes)<br />
Kinetics<br />
-Brain<br />
-Brain<br />
- Brain, DRG, s. cord<br />
- DRG, s. cord<br />
- Sk. muscle<br />
- DRG, brain, nodes<br />
-Heart<br />
- DRG (TTX R )<br />
- DRG (TTX R )<br />
Catterall et. al., 2003
Nav1.7 Mutations Cause Pain in Human<br />
Diseases<br />
Point Mutations<br />
Over-Active Channels<br />
S241T<br />
N395K<br />
I848T<br />
L858H,F<br />
A863P<br />
F1449V<br />
Primary Erythermalgia<br />
Paroxysmal Extreme<br />
Pain Disorder<br />
Nerve Hyperactivity<br />
Yang et al, 2004; Cummins et al, 2004 and others
Nav1.7 Loss of Function Causes Insensitivity<br />
to Pain<br />
I767X<br />
Chronic<br />
Insensitivity<br />
to Pain<br />
S459X<br />
Cox, J.J. et al, Nature 2006; Goldberg Y.P. et al., Clin. Genet. 2007
Cardiac Na + Channel Mutations<br />
Loss<br />
Gain<br />
Arrhythmia and<br />
Torsades de<br />
Pointes<br />
LQT and<br />
Torsades<br />
de Pointes
FLIPR-384: ΔVm Can Identify Blockers<br />
Na +<br />
12 known<br />
blockers<br />
A<br />
B<br />
C<br />
Na +<br />
-- channel<br />
-50mV<br />
opener<br />
Baseline<br />
fluorescence<br />
+ +<br />
+10mV<br />
Increased<br />
fluorescence<br />
Concentration (µM)<br />
0<br />
D<br />
E<br />
0.14<br />
F<br />
G<br />
0.41<br />
H<br />
I<br />
1.23<br />
J<br />
K<br />
3.70<br />
L<br />
M<br />
11.11<br />
N<br />
O<br />
33.33<br />
P<br />
100.00<br />
Range = ( -2000, 60000 )
Cell Supply
Blocker Characterization with <strong>IonWorks</strong> HT<br />
Propafenone blocks Nav in <strong>IonWorks</strong><br />
TTX<br />
Tetracaine<br />
Ambroxol<br />
Propafenone<br />
DMSO<br />
Vc (mV)<br />
-60 -40 -20 20 40<br />
-1<br />
Peak Current (nA)<br />
-2
Comparison of 23 standard Nav blockers<br />
Use-Dependence<br />
Selectivity<br />
100<br />
10<br />
20th IC50<br />
1st IC50<br />
100<br />
10<br />
Nav1.2<br />
Nav1.3<br />
Nav1.7<br />
Nav1.5<br />
Nav1.5 IC50 (µM)<br />
IC 50 (µM)<br />
1<br />
tetracaine<br />
ambroxol<br />
amitriptyline<br />
dibucaine<br />
(+/-)-fluoxetine<br />
mexiletine<br />
(+/-)-reboxetine<br />
riluzole<br />
vinpocetine<br />
lamotrigine<br />
lidocaine<br />
bupivacaine<br />
imipramine<br />
clomipramine<br />
propafenone<br />
haloperidol<br />
cinnarizine<br />
verapamil<br />
amiodarone<br />
S-(+)-fluoxetine<br />
(R,R)-reboxetine<br />
R-(-)-fluoxetine<br />
(S,S)-reboxetine<br />
-140 mV<br />
-20 mV<br />
P1 P20<br />
-70<br />
5-10 Hz<br />
1<br />
tetracaine<br />
ambroxol<br />
amitriptyline<br />
dibucaine<br />
(+/-)-fluoxetine<br />
mexiletine<br />
(+/-)-reboxetine<br />
riluzole<br />
vinpocetine<br />
lamotrigine<br />
lidocaine<br />
bupivacaine<br />
imipramine<br />
clomipramine<br />
propafenone<br />
haloperidol<br />
cinnarizine<br />
verapamil<br />
amiodarone<br />
S-(+)-fluoxetine<br />
(R,R)-reboxetine<br />
R-(-)-fluoxetine<br />
(S,S)-reboxetine
Correlation Between <strong>Assay</strong>s<br />
<strong>IonWorks</strong> HT vs. Manual EP<br />
<strong>IonWorks</strong> HT vs. FLIPR<br />
100<br />
3-fold boundary<br />
correlation fit, R 2 = 0.89<br />
1:1 fit<br />
1000<br />
100<br />
cinnarizine<br />
(clogP=6.1)<br />
mexiletine<br />
<strong>IonWorks</strong> HT IC 50<br />
(μM)<br />
10<br />
<strong>IonWorks</strong> IC 50<br />
(μM)<br />
10<br />
1<br />
(+/-)-reboxetine<br />
1<br />
1 10 100<br />
Manual Recording IC 50<br />
(μM)<br />
0.1<br />
Linear Fit, R 2 = 0.68<br />
1:1 correlation<br />
3 fold boundary<br />
TTX<br />
0.01<br />
0.01 0.1 1 10 100 1000<br />
FLIPR IC 50<br />
(μM)
Nav1.5 Block by Propafenone:<br />
IV relationship<br />
Control<br />
0.0<br />
Vc (mV)<br />
-40 0 40<br />
Propafenone 3 µM<br />
500 pA<br />
Current (nA)<br />
-0.5<br />
-1.0<br />
5 ms<br />
-1.5<br />
Control<br />
Propafenone 3µM
Nav1.5 Block by Propafenone:<br />
80<br />
60<br />
40<br />
20<br />
Use-dependent block & frequency-dependent IC 50<br />
3.9 µM<br />
Tonic<br />
Block<br />
Use-Dependent<br />
Block<br />
100<br />
0.5 Hz<br />
5 Hz<br />
1.0<br />
Normalized Current<br />
0.5<br />
0, 1 µM<br />
3 µM<br />
Block (%)<br />
1.4 µM<br />
10 µM<br />
0.0<br />
30 µM<br />
0 4 8 12<br />
Pulses<br />
Vh=-120mV<br />
0<br />
0.01 0.1 1 10 100<br />
Propafenone (μM)
Nav1.5 Block by Propafenone:<br />
Dose Response for holding potential of –120 and –80 mV<br />
100 V h<br />
=-120 mV<br />
80<br />
IC 50<br />
= 2.1 μM<br />
V h<br />
=-80 mV<br />
IC 50<br />
= 1μM<br />
Block (%)<br />
60<br />
40<br />
20<br />
-120 mV<br />
-80mV<br />
200ms<br />
-120mV<br />
-10 mV<br />
0<br />
n=4<br />
0.01 0.1 1 10 100<br />
Propafenone μM
Need for <strong>IonWorks</strong> <strong>Quattro</strong> <strong>Assay</strong><br />
n Greater throughput and data consistency (current size)<br />
2-4x increase in throughput<br />
n Allows single point screening from 96-well compound<br />
plate, while still maintaining replicates on 384-well<br />
plate<br />
Less cell line optimization needed
<strong>IonWorks</strong> <strong>Quattro</strong> <strong>Assay</strong><br />
Nav1.7<br />
I>200pA<br />
Expression Rate 95.3%<br />
Average peak current = 731 pA<br />
Average seal = 52.1 MΩ<br />
Gluconate in<br />
= 100 mM<br />
plate view<br />
I>200pA<br />
Nav1.5<br />
Expression Rate 97.7%<br />
Average peak current = 1184 pA<br />
Average seal = 30.4 MΩ<br />
Gluconate in<br />
= 100 mM
I-V curves of Nav1.7 and Nav1.5 on <strong>Quattro</strong><br />
Vc (mV)<br />
0.0<br />
-100 -80 -60 -40 -20 0 20<br />
-0.2<br />
Nav1,5 <strong>Quattro</strong><br />
0.0<br />
Vc (mV)<br />
-80 -60 -40 -20 0 20 40<br />
X Axis Title<br />
10 min after Amphotericin, n=366<br />
16 min after Amphotericin<br />
-0.4<br />
-0.6<br />
-0.8<br />
-1.0<br />
Nav1.7 peak current (nA)<br />
n=384<br />
Y Axis Title<br />
-0.5<br />
-1.0<br />
-1.5<br />
Nav1.5 peak current (nA)
Stability on <strong>IonWorks</strong> <strong>Quattro</strong><br />
Seal Stability<br />
Frequency (n)<br />
160<br />
140<br />
120<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
-30 -20 -10 -5 0 10 20 More<br />
Change in Resistance (%)<br />
Current Stability (6 min)<br />
30<br />
25<br />
Frequency (n)<br />
20<br />
15<br />
10<br />
90% cells: Run-up
Screening Nav1.7 State-Dependent Blockers<br />
using <strong>Quattro</strong><br />
Prepulse Vh = -140 mV<br />
Prepulse Vh = -70 mV<br />
Columns<br />
1 (R,R)-reboxetine<br />
2 dibucaine<br />
3 (+/-)-fluoxetine<br />
4 vinpocetine<br />
5 verapamil<br />
6 propafenone<br />
7 lamotrigine<br />
8 riluzole<br />
9 (S)-(-)bupivacaine<br />
10 imipramine<br />
11 tetracaine<br />
12 ambroxol<br />
10ms<br />
-20mV<br />
> 50% Block<br />
10ms<br />
-20mV<br />
3000ms<br />
-140mV<br />
3000ms<br />
-70mV<br />
-140mV<br />
50ms<br />
-140mV<br />
50ms
vinpocetine<br />
verapamil<br />
Tetrodotoxin<br />
tetracaine<br />
Standard 2<br />
standard 1<br />
100<br />
80<br />
60<br />
40<br />
20<br />
hNav1.7 Peak Current<br />
0<br />
IC 50<br />
2.9 µM at -140 mV<br />
IC 50<br />
1.1 µM at -70 mV<br />
120<br />
0.01 0.1 1 10 100<br />
µM<br />
100<br />
80<br />
60<br />
40<br />
20<br />
IC 50<br />
25.7 µM at -140 mV<br />
0<br />
IC 50<br />
11 µM at -70 mV<br />
0.01 0.1 1 10 100<br />
µM<br />
hNav1.7 Peak Current<br />
120<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
140<br />
120<br />
100<br />
80<br />
60<br />
40<br />
20<br />
State-dependent block of standard Nav<br />
blockers on <strong>Quattro</strong><br />
lamotrigine dibucaine<br />
10000<br />
-140 mV<br />
-70 mV<br />
IC 50<br />
>100 µM at -140 mV<br />
IC 50<br />
87.6 µM at -70 mV<br />
100<br />
0.1 1 10 100 1000<br />
µM<br />
propafenone<br />
verapamil<br />
1<br />
(+/-)-fluoxetine<br />
(+/-)-reboxetine<br />
(R,R)-reboxetine<br />
(S)-(-)bupivacaine<br />
ambroxol<br />
amiodarone<br />
cinnarizine<br />
clomipramine<br />
dibucaine<br />
haloperidol<br />
imipramine<br />
lamotrigine<br />
mexiletine<br />
propafenone<br />
R-(-)-fluoxetine<br />
IC 50<br />
9.9 µM at -140 mV<br />
IC 50<br />
8.1 µM at -70 mV<br />
0.01 0.1 1 10 100<br />
µM<br />
0.01<br />
1E-4<br />
hNav1.7 Peak Current<br />
hNav1.7 Peak Current<br />
IC50 (µM)<br />
riluzole<br />
S-(+)-fluoxetine<br />
0
Nav1.8 currents on <strong>IonWorks</strong><br />
<strong>IonWorks</strong> HT<br />
<strong>IonWorks</strong> <strong>Quattro</strong> (PPC)<br />
D7 Patch Plate<br />
0.5<br />
Scan #2<br />
0.4<br />
0.3<br />
0.2<br />
0.1<br />
Current (nA)<br />
0.0<br />
-0.1<br />
-0.2<br />
-0.3<br />
-0.4<br />
-0.5<br />
0 5 10 15 20 25<br />
Time (msec)<br />
I Na >100 pA<br />
Total<br />
Seals<br />
Number of Cells 28 290<br />
Expression Rate 10<br />
CsCl 140 mM and TTX 300 nM
Summary of Nav <strong>Assay</strong>s on <strong>IonWorks</strong><br />
n <strong>Quattro</strong> current expression rate can reach >95%, with<br />
peak currents >150 pA<br />
n Channel kinetics and pharmacology are consistent<br />
with manual patch<br />
n Platform provides EP data, use-dependence<br />
information, but in quantities similar to other platebased<br />
assays
Acknowledgements<br />
Neuroscience<br />
Howard Zhang<br />
Zhuangwei Lou<br />
Ed Kaftan<br />
Tony Lee<br />
John Dunlop<br />
Additional Thanks to:<br />
Qiang Lu (Novartis)<br />
Millipore (U.K.) Ion Channel group
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