Optimization of Ca(v)1.2 screening with an automated planar patch clamp platform.
BACKGROUND
Ca(v)1.2 channels play an important role in shaping the cardiac action potential. Screening pharmaceutical compounds for Ca(v)1.2 block is very important in developing drugs without cardiac liability. Ca(v)1.2 screening has been traditionally done using fluorescence assays, but these assays have some limitations. Patch clamping is considered the gold standard for ion channel studies, but is very labor intensive. The purpose of this study was to develop a robust medium throughput Ca(v)1.2 screening assay in PatchXpress 7000A by optimizing cell isolation conditions, recording solutions and experimental parameters. Under the conditions established, structurally different standard Ca(v)1.2 antagonists and an agonist were tested.


METHODS
HEK-293 cells stably transfected with hCa(v)1.2 L-type Ca channel were used. For experiments, cells were isolated using 0.05% Trypsin. Currents were recorded in the presence of 30 mM extracellular Ba2+ and low magnesium intracellular recording solution to minimize rundown. Ca(v)1.2 currents were elicited from a holding potential of -60 mV at 0.05 Hz to increase pharmacological sensitivity and minimize rundown. Test compounds were applied at increasing concentrations for 5 min followed by a brief washout.


RESULTS
Averaged peak Ca(v)1.2 current amplitudes were increased from 10 pA/pF to 15 pA/pF by shortening cell incubation and trypsin exposure time from 2.5 min at 37 degrees C to 1 min at room temperature and adding 0.2 mM cAMP to the intracellular solution. Rundown was minimized from 2%/min to 0.5%/min by reducing the intracellular free Mg2+ from 2.7 mM to 0.2 mM and adding 100 nM Ca2+. Under the established conditions, we tested 8 structurally different antagonists and an agonist. The IC(50) values obtained ranked well against published values and results obtained using traditional clamp experiments performed in parallel using the expressed cell line and native myocytes.


CONCLUSIONS
This assay can be used as a reliable pharmacological screening tool for Ca(v)1.2 block to assess compounds for cardiac liability during lead optimization.
Safety and Exploratory Pharmacology, Safety Assessment, Merck Research Laboratories, West Point, PA 19486, USA. bharathi_balasubramanian@merck.com
Bharathi
B
Balasubramanian
John P
JP
Imredy
David
D
Kim
Jacob
J
Penniman
Armando
A
Lagrutta
Joseph J
JJ
Salata
D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology*; D000818:Animals; D001464:Barium / Q000378:metabolism; D002120:Calcium Channel Agonists / Q000494:pharmacology*; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D020746:Calcium Channels, L-Type / Q000187:drug effects*; D002460:Cell Line; D017364:Collagenases / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation*; D004594:Electrophysiology; D006168:Guinea Pigs; D006352:Heart Ventricles / Q000166:cytology; D006801:Humans; D020128:Inhibitory Concentration 50; D007668:Kidney / Q000166:cytology; D008297:Male; D032383:Myocytes, Cardiac / Q000187:drug effects; D009543:Nifedipine / Q000494:pharmacology*; D018408:Patch-Clamp Techniques / Q000295:instrumentation* / Q000379:methods; D010447:Peptide Hydrolases / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors; D014162:Transfection; D014357:Trypsin / Q000494:pharmacology
D002120:Calcium Channel Agonists; D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type; D001464:Barium; D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; D010447:Peptide Hydrolases; D014357:Trypsin; D017364:Collagenases; D009543:Nifedipine