New PowerPoint - Aurora Biomed

Report
Biophysical and pharmacological characterization of
ligand-gated ion channels on QPatch in multi-hole mode
Rikke Schrøder
8th Ion Channel Retreat
29th of June, 2010
Outline
•
Introduction to QPatch
•
The multi-hole technology
•
Ligand-gated ion channels
on QPatch
•
Data
•
Summary
QPatch introduction
•
QPatch; a fully automated
industrial patch clamp
system
•
QPatch provides high
quality patch clamp data
based on giga seals
•
Current is continuously
recorded; real-time changes
are measured
•
Data analysis is automatic
and obtained with intuitive
and comprehensive QPatch
Assay Software
The QPatch family
•
QPatch 8/8X
•
QPatch 16/16X
•
QPatch HT/HTX
QPatch features
QPlate outside
•
Silicon-based patch clamp
orifice which replaces
conventional glass
pipettes
•
Exists in both single-hole
and multi-hole versions
•
Exists with either 16 or 48
measurement sites
QPlate inside
Upper Flow channel
•
•
•
•
•
Laminar flow
Multiple additions of compounds
Glass coated
5µl per liquid addition
Waste reservoir 70µl/250µl
QPatch multi-hole technology
•
The chip has multiple (10)
holes per well/measurement
site
•
The substrate is the silicon
chip; giga seals
•
Flow channels are the same;
laminar flow
•
The measured current is Itotal
•
Amplifier gain modifications;
measures current up to ±
100nA
Why multi-hole patching?
•
For assays with large cell-tocell heterogeneity in channel
expression
•
Transiently transfected cell lines
•
High consistency in data results
•
Nearly 100% success rate
•
Highest possible throughput
•
Lowest price per data point
Multi-hole challenges
•
Is the current rise time the
same for single-hole vs.
multi-hole experiments?
•
Is the biophysical/
pharmacological profile the
same for single-hole vs.
multi-hole experiments?
•
Is the success rate increased?
A
B
A
B
C
Ligand-gated applications on QPatch
•
Compounds can be applied
every 3 seconds (min. time
interval)
•
Liquid repetitions (e.g wash)
can be applied with intervals
of 1 second
•
Data acquisition and compound
application happen
simultaneously
•
Dose-response experiments
obtained on the same cell
•
Intelligent robot logic for
handling the pipettes for fast and
complex ligand-gated applications
Application protocol for 5 pt antagonist
dose-response experiments
47 liquid additions á 5 µl per experiment
ASIC1a Stability
ASIC1a recorded
at -60 mV
stimulated at pH
6.3
Reference; pH 6.3
Saline; pH 7.3
Minimum cycle duration 180 s gives
stable ASIC1a current respons
ASIC1a Rise time
ASIC1a Rise time (ms)
ASIC1a 10 – 90%
800
Rise time cursor (pH 6.3)
Single-hole data
pH 6.8
600
400
pH 6.3
ASIC1a 10 – 90%
200
Rise time cursor (pH 6.3)
Multi-hole data
0
pH 5.3
Multi-hole
Multi-hole
Multi-hole
Rise time data are the same for
Single-hole and multi-hole data
ASIC1a Desensitization
Single-mode
ASIC1a current
recorded at pH 6.3
Τavg=2812 ms ±
1783, n=8
Multi-mode
ASIC1a current
recorded at pH
6.3
Τavg=2101 ms
± 398, n=14
ASIC1a desensitizes within a few seconds in both single-hole and multi-hole
mode.
ASIC1a Pharmacology
ASIC1a current stimulated
with increasing
concentrations of protons
(pH 7.3, 6.8, 6.3, 5.3)
XC50 for known agonists/
antagonists (nM)
Protons
Single-
Multi-
Lit.
hole
hole
values
198
210
315
(pH 6.7)
(pH 6.7)
(pH 6.5)
Gründer,
Resulting concentration
response plot
EC50 = 210 nM
Chen 2010
% sites
with
ASIC
current
58
98
ASIC1a IV Single-hole
nA
0
-60
-20
-5
20
60
-10
-15
-20
-25
-30
Erev Na (avg) = 67 mV ± 9 mV,
n=4
Erev Na (theoretical) = 68 mV
ASIC1a IV Multi-hole
nA
0
-60
-20
20
60
-20
-40
-60
-80
-100
Erev Na (avg) = 73 mV ± 4 mV,
n=12
Erev Na (theoretical) = 68 mV
GABAA Rise time
10 – 90 % Rise time cursor
(12 µM GABA)
250
Single-hole data
GABAA Rise time (ms)
200
150
100
10 – 90 % Rise time cursor
50
(12 µM GABA)
Multi-hole data
0
12 µM
1
12 µM X-mode
Rise time for GABAA is similar
for single-hole vs. multi-hole
data
GABAA Pharmacology
XC50 for known
GABA
0.001
0.003
0.01
1
4 nA
0.03
250 ms
Leak subtracted
GABA α1β2γ2
currents in
response to
increasing
concentrations of
GABA (mM)
agonists/antagonists (µM)
GABA
Single-
Multi-
Lit.
hole
hole
values
5.6
12
9-18
Curtis et al,
0.1
1970
0.3
Bicuculline
1.2
1.5
1-3
Curtis et al,
1970
Normalized
GABA responses
fitted to the Hill
equation
EC50 = 12 µM
Diazepam
ND
0.14
0.15
@ 1 uM
Kapur et al.
GABA
1996
% sites
with current
37%
93%
GABAA Success rate
0
Single-hole
Multi-hole
37 %
93 %
-8.1
-39.2
+/- 8.1
+/- 22.3
10 uM GABA response (nA)
HT
% Sites with
-2000
GABA
current
-4000
HTX
-6000
Current
amplitude
(nA)
-8000
at 10 µM
GABA
-10000
Multi-hole technology increases
the success rate; from 37% to
93%
GABAA IV Single-hole
GABA current
stimulated with 10
µM GABA and
recorded at Vhold
-60, -40, -20, 0,
20, 40 mV
GABAA IV Multi-hole
GABA current
stimulated with 10
µM GABA and
recorded at Vhold
-60, -40, -20, 0,
20, 40 mV
GABAA IV
Multi-hole
Single-hole
nA
nA
-60
-40
25
25
20
20
15
15
10
10
5
5
0
-20 -5 0
20
40
-10
-60
-40
0
-20 -5 0
20
40
-10
Erev Cl (avg) = -38 mV ± 4 mV, n=6
Erev Cl (LJ corrected) = -51 mV
Erev Cl (avg) = -37 mV ± 3 mV, n=10
Erev Cl (LJ corrected) = -50 mV
Erev Cl (theoretical)
Erev Cl (theoretical)
= -51 mV
= -51 mV
GLuR5 Rise time multi-hole
GLuR5 10 – 90%
Rise time cursor
(GLuR5 activated by
0.3 mM kainate)
GLuR5 Rise time (ms)
250
200
150
100
GLuR5 10 – 90%
Rise time cursor
(GLuR5 activated by
3 mM kainate)
50
0
1
0.3 mM
3 mM
Rise time data for GLuR5 single
hole; N.D
GLuR5 Pharmacology
XC50 for known
GLuR5 currents
stimulated with 100 µM
kainate and blocked with
increasing concentrations
of CNQX
agonists/antagonists
(µM)
Glutamate
Single-
Multi-
Lit.
hole
hole
values
236
344
630
Lerma et al,
2001
Kainate
GLuR5 currents activated
wby 100 µM kainate in
increasing concentrations
of CNQX
IC50 = 3.6 µM
119
299
33-177
Lerma et al,
2001
CNQX
1.9
2.7
% sites with
65
91
Kainate
Rise time data are the same for
current
GLuR5 IV Multi-hole
nA
0.50
-60
-40
-20
0
20
-0.50
-1.50
-2.50
GLuR5 current activated by 3
mM kainate and recorded at
Vhold -60, -40, -20, 0, 20, 40
mV
Erev (avg) = 16 mV ± 10 mV,
n=14
40
TE671 nAChR alpha1 Rise time
α1 10 – 90% rise time cursor
(α1 activated by 10 mM ACh)
Multi-hole data
10 – 90 % rise time for α1 currents
Rise times are identical for the
two QPatch technologies
TE671 nAChR alpha1 Pharmacology
XC50 for known
α1 currents activated by
increasing conc. of ACh
(56 nM – 10 mM)
agonists/antagonists
(µM)
Acetyl
choline
Single-
Multi-
Lit.
hole
hole
values
5.6
8.1
8.5
Shao et al,
1998
Tetracaine
Grouped Hill fit for α1
currents activated with
ACh
EC50 = 8.1 µM
ND
1.6
13
Gentry &
Lukas, 2001
Gallamine
5.3
2.2
0.9
Poul et al,
2002
% sites
with current
90
93
TE671 nAChR alpha1 IV
X-mode
Single-mode
nA
nA
1
10
0.5
-60
0
-10
-0.5
-1
-1.5
5
40
-60
0
-10
-5
-10
-15
-20
Erev (avg) = 8 mV ± 2 mV, n=5
Erev (avg) = 8 mV ± 3 mV, n=11
Erev (LJ corrected) = -5 mV
Erev (LJ corrected) = -5 mV
40
Screening on ligand-gated ion channels
New feature: Screening on ligand-gated ion
channels is possible using the next software version
from Sophion; will be released Fall 2010
Summary
Using X-mode technology
•
Is the signal onset slower?
•
Is the biophysical/
pharmacological profile
correct?
•
Is the success rate increased?
Summary; Rise time data
The current rise time from
different ligand gated ion
channels is comparable for
multi-hole and single-hole
data
It is assumed that the angle
of the liquid front in the flow
channel ”hits the cell” from
above rather than from the
side
Summary
Using X-mode technology
•
Is the signal onset slower?
•
Is the biophysical/
pharmacological profile
correct?
•
Is the success rate
increased?
Summary
Using X-mode technology
•
•
•
% Sites with current
Singlehole
Multihole
ASIC1a
58
98
GABAA
37
93
GLuR5
65
91
nAChα1
90
93
Is the signal onset slower?
Is the the biophysical/
pharmacological profile
correct?
Is the success rate
increased?
Acknowledgement
X-mode team
Rasmus Bjørn Jacobsen
Simon Pedersen
Jesper Gerved
Jens Henneke
Patrick Juhl
Jonatan Kutchinsky
Morten Sunesen
Søren Friis
Ligand data
Søren Friis
Hervør Lykke Olsen
Knirke Jensen
Rasmus Bjørn Jacobsen
Dorthe Nielsen
Mette Christensen
Jeffery Weber
For more information
WWW.SOPHION.COM

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