Automated Patch-Clamping. Presentation at the Nanion Usergroup Meeting, Munich, Sept 2009

1. Round Table Discussion
Thoughts on APC assay optimization -
Strengths and weaknesses of APC instruments
Prof. Clemens Möller, PhD
Albstadt-Sigmaringen University of Applied Sciences
office@biophysicalconsulting.de
www.clemensmoller.de
Nanion Usergroup Meeting
Sept 29, 2011

2. Correlation MPC-APC
Assay set up
Flexibility of operation;
primary cells
2
Round Table Discussion

3. Controlled
state of
channel
Low binding
of
hydrophobic
compounds
Low leak
currents
Control of
membrane
potential /
capacitance
Patch-
Clampers
desire to
tinker with
the
experiment
PatchLiner, Port-a-Patch & SyncroPatch
data are in excellent correlation to MPC
Continuous
voltage
control
Chips made of
glass
substrate
Gigaseals
RS
compensation
HEKA
Software for
experiment
and data
evaluation
3
Experiments are performed under similar conditions as in MPC

4. Controlled
state of
channel
Low binding
of
hydrophobic
compounds
Low leak
currents
Control of
membrane
potential /
capacitance
Patch-
Clampers
desire to
tinker with
the
experiment
PatchLiner, Port-a-Patch & SyncroPatch
data are in excellent correlation to MPC
Continuous
voltage
control
Chips made of
glass
substrate
Gigaseals
RS
compensation
HEKA
Software for
experiment
and data
evaluation
4
Experiments are performed under similar conditions as in MPC
Main difference to MPC: Cells are delivered from suspension (not adherent).
 Pharmacology? Networks of cells?

5. Potential
(mV)
-100 -50 0
-2
-1
0
1
Peak current
(nA)
.
potential (mV)
-140 -120 -100 -80 -60 -40 -20 0 20 40
current(nA)
-3.5
-3.0
-2.5
-2.0
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
.
Biophysical characterization of hERG channels
5
Manual Patch-Clamp
PatchLiner
Current-voltage relationships of hERG channels correlate very well
Reference: Möller and Witchel (submitted).

6. 6
Excellent correlation between manual and planar patch clamp
Before employing automated (planar)
patch-clamping in our programs, the
devices were validated with reference
and actual program compounds.
57 compounds within one chemical
series were tested side by side on
manual rigs and a planar patch clamp
device (PatchLiner)
Only 5 out of 57 compounds showed a
difference in the IC50-values of ~5-fold
Correlation: Manual Patch-Clamp vs PatchLiner
Manual Patch-Clamp IC50 [µM]
0.1 1 10 100
PatchLinerIC50[µM]
0.1
1
10
100
5
52
Pharmacological comparison of
Electrophysiology Platforms
PatchLiner validation
Reference: Davenport et al., 2010

7. Key points for pharmacology: same as for MPC and HT systems
o Prepare compound solutions as freshly as possible. Observe solubility of
compounds.
o Compound stock solutions required? How long are the compounds stable in
DMSO, at which storage temperature?
o Store solutions with reduced vehicle (DMSO) content in glassware, for as
short period as possible.
o Are currents stable under negative control conditions? Any vehicle effects?
o Do currents reach steady state in presence of compounds? (No continuous
perfusion in APC; repeated cpd administration required?)
o Consider pulse protocols (do the compounds exhibit preference for certain
states of the channel?)
o Consider temperature effects
7
PatchLiner, Port-a-Patch & SyncroPatch
data are in excellent correlation to MPC

8. (Many) Port-A-Patch, PatchLiner and
SyncroPatch assays are easy to set up
o Operation in standard modes easy
o Very "forgiving" cell culture
o Good seal success rates can be achieved with
suboptimal cells
o But: Seal enhancer (for most cell types)
appears to be required for good success
rates?
8
Eccellent seal success rates
Kv1.5
HERG
1st tier profilingExample: Panel of cardiac channels on PL
Kv1.1
Kv4.3/KChIP2
NaV1.5
L-type Ca2+
Standard protocols for most cell types
and channels are available.
For many cells, excellent success rates
can be achieved.
Current traces from Möller et al., 2010

9. (Many) Port-A-Patch, PatchLiner and
SyncroPatch assays are easy to set up
o The healthier the cells are, the better the seal success rate will typically be
o Cell confluency ~60-80%. Can depend on cell type
o Especially small / large cells?  Consider different chip hole size
o Cell density appears to be not so critical (1 x 106 – 5 x 107 cells/ml are good
standard densities, but much lower densities have worked fine for some cells)
o Relatively small effect of pressure etc. settings in PatchControl software; standard
settings are often a good choice
9
Key points to consider for a good seal success rate

10. APC instruments complement each other
In addition, the Port-A-Patch proved very useful for
o Assay development support for PatchLiner and SyncroPatch
o Verification of data for compounds that showed inconsistent IC50 values on
the PatchLiner or the SyncroPatch
10
Instruments for different needs of throughput
Port-a-Patch PatchLiner SyncroPatch

11. APC instruments are highly flexible
11
Recordings from primary cells possible
300 nM Haloperidol
Negative control
Neurons – MAP2
astrocytes – GFAP
Nuclei – DRAQ5
Neurons Cardiomyocytes
Na+
Ca2+
K+
Reference: Möller et al., 2010

12. Modes of operation
12
Excised patch recordings not (yet, really) possible by planar APC
"Whole-
cell"
• Most widely used for pharmacology
"Cell-
attached
"
• Possible with some cells
"excised
patch"
• Are you missing single channel recordings
by APC? For MoA?

13. o Different features available
o Voltage clamp, current clamp (action potential recordings)
o Whole cell, perforated patch (are you using this a lot?)
o Intracellular solution exchange
o Fast ligand exchange (~50 ms)
o Temperature control
o Interaction during experiment possible
o Patch-Clampers desire to "play around" with an experiment
o Also, a "screening mode" with limited user access to settings is possible
(Talk by Corinna from last years meeting)
Different features available
APC instruments are highly flexible
13
Many features available; interaction possible
Interaction during experiment possible

14. Thank you for your attention and input!
Andreas Ebneth
Rainer Netzer
Heike Deisemann
Desireé Amm
York Rudhard
John Kemp
The whole great team,
especially:
Niels, Andrea, Michael,
Claudia, Sonja, Timo, Ali &
Cecilia
Ralf Kettenhofen
Martin Stolz
Prof. Clemens Möller, PhD | Albstadt-Sigmaringen University of Applied Sciences
clemens@biophysicalconsulting.de | www.clemensmoller.de
14
Carsten Claussen
Clemens Möller | Albstadt-Sigmaringen University of Applied Sciences
office@biophysicalconsulting.de | www.clemensmoller.de

15. References & Recommended Reading
15
References:
- Clemens Möller (2010). Keeping the Rhythm: hERG and beyond in Cardiovascular Safety Pharmacology. Expert Reviews Clinical
Pharmacology (Ion Channels) 3: 3. 321-329 May
- Adam J Davenport, Clemens Möller, Alexander Heifetz, Michael P Mazanetz, Richard J Law, Andreas Ebneth, Mark J Gemkow
(2010). Using Electrophysiology and in silico 3D Modelling to reduce hERG inhibition in a Histamine H3 Receptor Antagonist Program.
ASSAY and Drug Development Technologies 8: 6. 781-789 December
- Clemens Möller, Mark Slack (2010). Impact of new technologies for cellular screening along the drug value chain. Drug Discovery
Today 15: 9-10. 384-390 May
- Clemens Möller, Harry Witchel. Automated Electrophysiology Makes the Pace for Cardiac Ion Channel Safety Screening. (Submitted to
Frontiers, 2011)
Recommended reading:
Carol J Milligan, Li J, Sukumar P, Majeed Y, Dallas ML, English A, Emery P, Porter KE, Smith AM, McFadzean I, Beccano-Kelly
D, Bahnasi Y, Cheong A, Naylor J, Zeng F, Liu X, Gamper N, Jiang LH, Pearson HA, Peers C, Robertson B, Beech DJ (2009). Robotic
multiwell planar patch-clamp for native and primary mammalian cells. Nat Protoc. 2009;4(2):244-55.