Faculty Collaboration Database

Characterization of GABA_A receptor ligands with automated patch-clamp using human neurons derived from pluripotent stem cells. J Pharmacol Toxicol Methods 2016;82:109-114

Date

10/18/2016

Pubmed ID

27544543

Pubmed Central ID

PMC5116268

DOI

10.1016/j.vascn.2016.08.006

Scopus ID

2-s2.0-84989944608 (requires institutional sign-in at Scopus site)   10 Citations

Abstract

INTRODUCTION: 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 GABA_A receptor.

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 GABA_A receptor modulators. Both efficacy and affinity were determined. In addition, mRNA of GABA_A receptor 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 EC₅₀ of 0.43μM. Positive GABA_A receptor modulators diazepam, HZ-166, and CW-04-020 exhibited EC₅₀ values 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 GABA_A receptor 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.

DISCUSSION: iCell Neurons represent a new cellular model to characterize GABAergic compounds using automated patch clamp. These cells have excellent representation of cellular GABA_A receptor distribution that enable determination of total small molecule efficacy and affinity as measured by cell membrane current change.

Author List

Yuan NY, Poe MM, Witzigmann C, Cook JM, Stafford D, Arnold LA

Authors

Alexander (Leggy) Arnold PhD Professor in the Chemistry & Biochemistry department at University of Wisconsin - Milwaukee
James M. Cook PhD University Distinguished Professor in the Chemistry and Biochemistry department at University of Wisconsin - Milwaukee

MESH terms used to index this publication - Major topics in bold

Allosteric Regulation
Cell Culture Techniques
Cells, Cultured
GABA-A Receptor Agonists
Humans
Ligands
Membrane Potentials
Neurons
Patch-Clamp Techniques
Pluripotent Stem Cells
Protein Subunits
Receptors, GABA-A