title: Utility of frozen cell lines in medium throughput electrophysiology screening of hERG and NaV1.5 blockade.
abstract: BACKGROUND
  The development of drug candidates must take into account that many compounds have off-target activity against voltage-gated ion channels (VGIC) which may prevent their progression to market. Of particular concern are hERG and hNa(V)1.5. Screening against these ion channels is necessary but expensive, partially due to maintenance of constantly cultured cell lines. Here, we show that frozen HEK-293 cells can be maintained indefinitely, reducing variability in cell performance, time and expense of cell culture.
  
  
  METHODS
  Cells, constantly cultured or frozen, were assayed on the PatchXpress 7000A using tool compounds.
  
  
  RESULTS
  Amitriptyline, quinidine, compound A, fluoxetine and imipramine inhibited hERG with IC(50)s (paired values denote constantly cultured and frozen, respectively) of 4.8±0.4 and 5.1±0.4, 1.4±0.1 and 1.1±0.1, 24.4±2.4 and 21.9±1.8, 2.1±0.4 and 2.1±0.1, 5.2±0.4 and 4.0±0.2μM. Quinidine, flecainide, mexiletine and amitriptyline inhibited hNa(V)1.5 with IC(50)s of 46.6±4.3 and 28.0±2.3, 7.6±0.7 and 6.2±0.5, 153.5±13.0 and 106.0±4.7, 5.5±0.5 and 4.8±0.2μM. Voltage dependences of activation (V(1/2)) for hERG were statistically identical, 0.4±0.8mV and 2.5±0.5mV. In hNa(V)1.5, the V(1/2) of inactivation and activation were statistically identical, -82.7±0.1mV versus -84.9±0.3mV, -47.5±0.3mV versus -45.0±0.6mV. Current density in both conditions in hERG experiments was similar, 47.0±4.1pA versus 42.3±6.0pA/pF.
  
  
  CONCLUSIONS
  hERG and hNa(V)1.5 screens run using frozen cells have statistically identical IC(50)s, voltage dependence of activation, IV relationships and current density to screens using continuously cultured cells. Frozen cells have more constant performance and allow rapid switching between experiments on several cell lines without sacrificing data quality.
authors: Screening and Compound Profiling, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, USA. brian.t.donovan@gsk.com
authors: Brian T
authors: BT
authors: Donovan
authors: Tania
authors: T
authors: Bakshi
authors: Sarah E
authors: SE
authors: Galbraith
authors: Christopher J
authors: CJ
authors: Nixon
authors: Lisa A
authors: LA
authors: Payne
authors: Stan F
authors: SF
authors: Martens
mesh_terms: D000639:Amitriptyline / Q000494:pharmacology; D002461:Cell Line, Transformed; D015925:Cryopreservation / Q000379:methods; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D000072237:ERG1 Potassium Channel; D055724:Electrophysiological Phenomena / Q000187:drug effects*; D051638:Ether-A-Go-Go Potassium Channels / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D005473:Fluoxetine / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D007099:Imipramine / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects; D062554:NAV1.5 Voltage-Gated Sodium Channel; D018408:Patch-Clamp Techniques / Q000379:methods; D026902:Potassium Channel Blockers / Q000494:pharmacology*; D011802:Quinidine / Q000494:pharmacology; D015222:Sodium Channels / Q000378:metabolism*
chemical_list: D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; C000606913:KCNH2 protein, human; D062554:NAV1.5 Voltage-Gated Sodium Channel; D026902:Potassium Channel Blockers; C568320:SCN5A protein, human; D015222:Sodium Channels; D005473:Fluoxetine; D000639:Amitriptyline; D011802:Quinidine; D007099:Imipramine