title: Characterization of GABAAreceptor ligands with automated patch-clamp using human neurons derived from pluripotent stem cells.
abstract: BACKGROUND
  Automated patch clamp is a recent but widely used technology to assess pre-clinical drug safety. With the availability of human neurons derived from pluripotent stem cells, this technology can be extended to determine CNS effects of drug candidates, especially those acting on the GABAAreceptor.
  
  
  METHODS
  iCell Neurons (Cellular Dynamics International, A Fujifilm Company) were cultured for ten days and analyzed by patch clamp in the presence of agonist GABA or in combination with positive allosteric GABAAreceptor modulators. Both efficacy and affinity were determined. In addition, mRNA of GABAAreceptor subunits were quantified by qRT-PCR.
  
  
  RESULTS
  We have shown that iCell Neurons are compatible with the IonFlux microfluidic system of the automated patch clamp instrument. Resistance ranging from 15 to 25MΩ was achieved for each trap channel of patch clamped cells in a 96-well plate format. GABA induced a robust change of current with an EC50of 0.43μM. Positive GABAAreceptor modulators diazepam, HZ-166, and CW-04-020 exhibited EC50values of 0.42μM, 1.56μM, and 0.23μM, respectively. The α2/α3/α5 selective compound HZ-166-induced the highest potentiation (efficacy) of 810% of the current induced by 100nM GABA. Quantification of GABAAreceptor mRNA in iCell Neurons revealed high levels of α5 and β3 subunits and low levels of α1, which is similar to the configuration in human neonatal brain.
  
  
  CONCLUSIONS
  iCell Neurons represent a new cellular model to characterize GABAergic compounds using automated patch clamp. These cells have excellent representation of cellular GABAAreceptor distribution that enable determination of total small molecule efficacy and affinity as measured by cell membrane current change.
authors.affiliation: Department of Chemistry and Biochemistry and Milwaukee Institute for Drug Discovery, University of Wisconsin - Milwaukee, 3210 N. Cramer Street, Milwaukee, WI 53211, United States. Electronic address: ninayuan@uwm.edu.
authors.forename: Nina Y
authors.initials: NY
authors.lastname: Yuan
authors.affiliation: Department of Chemistry and Biochemistry and Milwaukee Institute for Drug Discovery, University of Wisconsin - Milwaukee, 3210 N. Cramer Street, Milwaukee, WI 53211, United States. Electronic address: mmpoe@uwm.edu.
authors.forename: Michael M
authors.initials: MM
authors.lastname: Poe
authors.affiliation: Department of Chemistry and Biochemistry and Milwaukee Institute for Drug Discovery, University of Wisconsin - Milwaukee, 3210 N. Cramer Street, Milwaukee, WI 53211, United States. Electronic address: witzigm2@uwm.edu.
authors.forename: Christopher
authors.initials: C
authors.lastname: Witzigmann
authors.affiliation: Department of Chemistry and Biochemistry and Milwaukee Institute for Drug Discovery, University of Wisconsin - Milwaukee, 3210 N. Cramer Street, Milwaukee, WI 53211, United States. Electronic address: capncook@uwm.edu.
authors.forename: James M
authors.initials: JM
authors.lastname: Cook
authors.affiliation: Department of Chemistry and Biochemistry and Milwaukee Institute for Drug Discovery, University of Wisconsin - Milwaukee, 3210 N. Cramer Street, Milwaukee, WI 53211, United States. Electronic address: dcstaff@uwm.edu.
authors.forename: Douglas
authors.initials: D
authors.lastname: Stafford
authors.affiliation: Department of Chemistry and Biochemistry and Milwaukee Institute for Drug Discovery, University of Wisconsin - Milwaukee, 3210 N. Cramer Street, Milwaukee, WI 53211, United States. Electronic address: arnold2@uwm.edu.
authors.forename: Leggy A
authors.initials: LA
authors.lastname: Arnold
mesh_terms: D000494:Allosteric Regulation; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D058785:GABA-A Receptor Agonists / Q000494:pharmacology*; D006801:Humans; D008024:Ligands; D008564:Membrane Potentials / Q000187:drug effects; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D018408:Patch-Clamp Techniques; D039904:Pluripotent Stem Cells / Q000166:cytology*; D021122:Protein Subunits; D011963:Receptors, GABA-A / Q000235:genetics / Q000378:metabolism*
chemical_list: D058785:GABA-A Receptor Agonists; D008024:Ligands; D021122:Protein Subunits; D011963:Receptors, GABA-A
keywords: CW-04-020; Diazepam; GABA; GABA(A) receptor; HZ-166; Human-induced pluripotent stem cell; Ionflux; Patch clamp; iCell neuron