Characterization of GABA_A receptor ligands with automated patch-clamp using human neurons derived from pluripotent stem cells

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• 作者：Nina Y. Yuan ^{ninayuan@uwm.edu" class="auth_mail" title="E-mail the corresponding author} ; Michael M. Poe ^{mmpoe@uwm.edu" class="auth_mail" title="E-mail the corresponding author} ; Christopher Witzigmann ^{witzigm2@uwm.edu" class="auth_mail" title="E-mail the corresponding author} ; James M. Cook ^{capncook@uwm.edu" class="auth_mail" title="E-mail the corresponding author} ; Douglas Stafford ^{dcstaff@uwm.edu" class="auth_mail" title="E-mail the corresponding author} ; Leggy A. Arnold ; ^{arnold2@uwm.edu" class="auth_mail" title="E-mail the corresponding author}
• 关键词：CW-04-020 ; Diazepam ; GABA ; GABAA receptor ; Human-induced pluripotent stem cell ; HZ-166 ; iCell neuron ; Ionflux ; Patch clamp
• 刊名：Journal of Pharmacological and Toxicological Methods
• 出版年：2016
• 出版时间：November-December 2016
• 年：2016
• 卷：82
• 期：Complete
• 页码：109-114
• 全文大小：628 K

文摘

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 25 MΩ 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 0.43 μM. Positive GABA_A receptor modulators diazepam, HZ-166, and CW-04-020 exhibited EC₅₀ values of 0.42 μM, 1.56 μM, and 0.23 μM, respectively. The α2/α3/α5 selective compound HZ-166-induced the highest potentiation (efficacy) of 810% of the current induced by 100 nM 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.