nanion usergroup meeting sept 2011
DESCRIPTION
Automated Patch-Clamping. Presentation at the Nanion Usergroup Meeting, Munich, Sept 2009TRANSCRIPT
Round Table Discussion
Thoughts on APC assay optimization -
Strengths and weaknesses of APC instruments
Prof. Clemens Möller, PhDAlbstadt-Sigmaringen University of Applied [email protected]
Nanion Usergroup Meeting
Sept 29, 2011
· Correlation MPC-APC
· Assay set up
· Flexibility of operation;
primary cells
2
Round Table Discussion
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
substrateGigaseals RS
compensation
HEKA Software for experiment
and data evaluation
3
Experiments are performed under similar conditions as in MPC
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
substrateGigaseals 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?
Potential(mV)
-100 -50 0
-2
-1
0
1
Peak current(nA)
.
potential (mV)
-140 -120 -100 -80 -60 -40 -20 0 20 40
curr
en
t (n
A)
-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
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
Pat
chL
iner
IC
50
[µM
]
0.1
1
10
100
5
52
Pharmacological comparison of Electrophysiology Platforms
PatchLiner validation
Reference: Davenport et al., 2010
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
(Many) Port-A-Patch, PatchLiner and SyncroPatch assays are easy to set up
o Operation in standard modes easy
o Very "forgiving" cell cultureo 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 ratescan be achieved.
Current traces from Möller et al., 2010
(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 beo Cell confluency ~60-80%. Can depend on cell type o Especially small / large cells? Consider different chip hole sizeo 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
APC instruments complement each other
In addition, the Port-A-Patch proved very useful foro Assay development support for PatchLiner and SyncroPatcho 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
APC instruments are highly flexible
11
Recordings from primary cells possible
300 nM Haloperidol
Negative control
Neurons – MAP2astrocytes – GFAP
Nuclei – DRAQ5
Neurons Cardiomyocytes
Na+
Ca2+
K+
Reference: Möller et al., 2010
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?
??
o Different features availableo Voltage clamp, current clamp (action potential recordings)o Whole cell, perforated patch (are you using this a lot?)o Intracellular solution exchangeo Fast ligand exchange (~50 ms)o Temperature control
o Interaction during experiment possible
o Patch-Clampers desire to "play around" with an experimento 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
Thank you for your attention and input!
Andreas EbnethRainer NetzerHeike DeisemannDesireé AmmYork RudhardJohn 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 [email protected] | www.clemensmoller.de
14
Carsten Claussen
Clemens Möller | Albstadt-Sigmaringen University of Applied Sciences
[email protected] | www.clemensmoller.de
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.