Nav1.5 on IonWorks HT - Molecular Devices

Compound Screening against Cardiac
Sodium Channels with IonWorks TM HT
Using a Native Cell Line
Qiang Lü, Ph.D.
Principal Research Scientist
Discovery Neuroscience
Feb. 10, 2005

Outline
n Sodium Channels: Drug Target and Liabilities
n RT4-B8 Cells
Origin
<strong>Nav1.5</strong> in RT4-B8
n IonWorks HT Application Studies
Basic Characterization
Pharmacology Studies
Use- and State- Dependence
n Summary

Voltage-gated Na + (Nav) Channels are
Molecular Targets for Various Indications
n Na + influx generates and propagates action potentials
n Modulation of Na + current affects AP in excitable cells
n Nav channel blockers have been used for various
indications
- Including local anesthetics, antiarrhythmics, anticonvulsants, and
antidepressant
- Non-selectivity among Nav channels causes adverse side effects
n Need selectivity for validated Nav channel targets
- avoiding cardiac Nav channel(s) for safety

Family of Nav Channels
Yu and Catterall, 2003
Heart
nM TTX-resistant
Catterall et al, 2003

RT4-B8 Has Endogenously Expressed <strong>Nav1.5</strong>
Northern Blot:
n
n
n
Source: derived from a multipotent
stem cell line, RT4-AC, which
originated from a rat peripheral
neurotumor
Overall sequence identity between
r<strong>Nav1.5</strong> and h<strong>Nav1.5</strong>: 94.2%
Sequence identity within fourrepeat
area: 98.9%
Zeng, et al, 1996

RT4-B8 Na Current Pharmacology: TTX
Sensitivity
20x
20x
-120mV
120
5Hz
-120mV
cpd 3-5 min
-10mV
10ms
20x
% Inhibition
100
80
60
40
20
5 Hz
IC50=1.4 µM
0
0.01 0.1 1 10 100 1000 10000
[TTX] (µM)

RT4-B8 Na Current Electrophysiology
Vc (mV)
-60 -40 -20 0 20 40 60
0.0
1.0
Current (nA)
-0.5
-1.0
Normalized Current
0.5
-1.5
0.0
n=9
-150 -100 -50
V Pre
(mV)

FLIPR Comparison of h<strong>Nav1.5</strong> and r<strong>Nav1.5</strong> in
RT4-B8
100
IC 50
(FLIPR h<strong>Nav1.5</strong>) (µM)
10
R 2 = 0.83
1
1 10 100
IC 50
(FLIPR RT4-B8) (µM)

IonWorks HT: An Automated E-phys Platform
n Hardware
384-well based cell plate
48-head parallel recording
- Same voltage protocol for 48-head
- Cells are clamped only during application of
voltage protocol
Compound addition (one time)
Compound source: 96-well plate
n Software and Data Analysis
Linear leak subtraction without P/N
Flexible metric setting and export function
In-house software for data formatting and
analysis

RT4-B8 Yielded High Success Rate for
IonWorks HT:
RT4-B8
HEK293H/<strong>Nav1.5</strong> Clone 219
RT4-B8
HEK/Clone 219
Seal
187/192 (-5)
97.4%
170/192 (-22)
88.5%
Seal

<strong>Nav1.5</strong> on IonWorks HT:
Success Rate on Various Runs
400
2000
Mean Number per Plate
300
200
100
Mean Current per Cell (nA)
1000
0
Seals Current Stable Current
0
1
RT4-B8 from 5 plates on various days

A Representative Plate

<strong>Nav1.5</strong> on IonWorks HT:
Current Size Distribution
C6 Patch Plate
Scan #1
1
60
0
Number of Cells
40
20
I mean
= 1.51nA
I med
= 1.20nA
SD = 1.11nA
SEM = 0.06nA
N = 335
Current (nA)
-1
-2
-3
-4
-5
-6
5 10 15 20 25 30
Time (msec)
0
0 1 2 3 4 5 6
Current (nA)

<strong>Nav1.5</strong> on IonWorks HT: Stability Test
4-hr Cell 2-hr Cell 1-hr Cell
4-hr cell 2-hr cell 1-hr cell
% seals from 128: 84.4 92.2 90.6
% currents(pre) remaining after filters, from seals: 55.6 83.1 81.0
% stable currents (-Stability%

I-V Curve: IonWorks HT vs Patch-clamp
IonWorks HT
Whole-cell Patch-clamp
Vc (mV)
-60 -50 -40 -30 -20 -10 0 10 20 30 40 50
0.0
Vc (mV)
-60 -40 -20 0 20 40 60
0.0
-0.5
Normalized I
Current (nA)
-0.5
-1.0
-1.0
-1.5

Voltage Dependent Inactivation of Nav
Channels in RT4-B8 by Manual Patch-Clamp
-10 mV
10 ms
1.0
-55 mV
-140 mV
3 Sec
Control <strong>Nav1.5</strong>
-120 mV
Normalized Current
0.5
2nA
25 msec
0.0
n=9
-150 -100 -50
V Pre
(mV)

<strong>Nav1.5</strong> on IonWorks HT:
Voltage Protocol and Metric Determination

<strong>Nav1.5</strong> on IonWorks HT:
Plate View of Tetracaine Blocking <strong>Nav1.5</strong>
>10% block
>50% block >95% block
Increasing
Doses

Example Current Traces for Compound Well
G8

<strong>Nav1.5</strong> on IonWorks HT:
Consistency of Dose Responses
% Current
140
120
100
80
60
40
20
0
0.001 0.01 0.1 1 10 100 1000
IC50 = 8.6µM amitriptyline, cpd-plate columns 1-3
µM
% Current
160
140
120
100
80
60
40
20
0
0.001 0.01 0.1 1 10 100 1000
IC50 = 14.3µM imipramine, cpd-plate columns 4-6
µM
% Current
160
140
120
100
80
60
40
20
0
0.001 0.01 0.1 1 10 100 1000
IC50 = 12.4µM amitriptyline, cpd-plate columns 7-9
µM
% Current
160
140
120
100
80
60
40
20
0
0.001 0.01 0.1 1 10 100 1000
IC50 = 18.8µM imipramine, cpd-plate columns 10-12
µM

IC 50 Measurement: Correlation between
IonWorks HT and Patch-Clamp with RT4-B8
100
IC 50
(IonWorks) (µM)
10
R 2 = 0.78
1
1 10 100
IC 50
(e-phys) (µM)

<strong>Nav1.5</strong> on IonWorks HT:
Use-Dependent Block of <strong>Nav1.5</strong>
First Pulse
8 th Pulse
33.3µM compound A

<strong>Nav1.5</strong> on IonWorks HT:
State-Dependent Block of <strong>Nav1.5</strong>
Normalized Current
1.0
0.5
0.0
n=9
-150 -100 -50
V Pre
(mV)
% current
160
140
120
100
80
60
40
20
0
0.001 0.01 0.1 1 10 100 1000
uM
IC50 = 30.1µM ambroxol, V H = -150mV
% current
160
140
120
100
80
60
40
20
0
0.001 0.01 0.1 1 10 100 1000
uM
IC50 = 11.4µM ambroxol, V H = -100mV
V H = -150mV vs. V H = -100mV

Summary
n
n
n
RT4-B8 is a native cell line that endogenously express <strong>Nav1.5</strong>
without appearance of other Nav currents
Northern and RT-PCR
Characterization by electrophysiology and pharmacology
RT4-B8 is “IonWorks HT friendly”
Seal rate
Current size
Current stability
Easy for manual patch-clamp
IonWorks HT can be used on RT4-B8 to study <strong>Nav1.5</strong> compounds
IC 50 measurement
Correlation with patch-clamp
Use-dependent
State-dependent

Acknowledgements
n
Wyeth Research (co-authors)
Howard Zhang
Ru Shen
Lan He
Mark Bowlby
John Dunlop
Ed Kaftan
Steve Lin
Brian Jow
n
n
Molecular Devices Inc.
Fred Livingston
Sue Murphy
University of Chicago
Dorothy Hanck