Ionotropic GABA and glycine receptor subunit composition in human pluripotent stem cell-derived excitatory cortical neurones

Owain T. James, Matthew R. Livesey, Jing Qiu, Owen Dando, Bilada Bilican, Ghazal Haghi, Rinku Rajan, Karen Burr, Giles E. Hardingham, Siddharthan Chandran, Peter C. Kind, David J. A. Wyllie*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

We have assessed, using whole-cell patch-clamp recording and RNA-sequencing (RNA-seq), the properties and composition of GABA(A) receptors (GABA(A)Rs) and strychnine-sensitive glycine receptors (GlyRs) expressed by excitatory cortical neurons derived from human embryonic stem cells (hECNs). The agonists GABA and muscimol gave EC50 values of 278 mu M and 182 mu M, respectively, and the presence of a GABA(A)R population displaying low agonist potencies is supported by strong RNA-seq signals for alpha 2 and alpha 3 subunits. GABA(A)R-mediated currents, evoked by EC50 concentrations of GABA, were blocked by bicuculline and picrotoxin with IC50 values of 2.7 and 5.1 mu M, respectively. hECN GABA(A)Rs are predominantly gamma subunit-containing as assessed by the sensitivity of GABA-evoked currents to diazepam and insensitivity to Zn2+, together with the weak direct agonist action of gaboxadol; RNA-seq indicated a predominant expression of the gamma 2 subunit. Potentiation of GABA-evoked currents by propofol and etomidate and the lack of inhibition of currents by salicylidine salycylhydrazide (SCS) indicate expression of the beta 2 or beta 3 subunit, with RNA-seq analysis indicating strong expression of beta 3 in hECN GABAARs. Taken together our data support the notion that hECN GABA(A)Rs have an alpha 2/3 beta 3 gamma 2 subunit composition - a composition that also predominates in immature rodent cortex. GlyRs expressed by hECNs were activated by glycine with an EC50 of 167 mu M. Glycine-evoked (500 mu M) currents were blocked by strychnine (IC50 = 630 nM) and picrotoxin (IC50 = 197 mu M), where the latter is suggestive of a population of heteromeric receptors. RNA-seq indicates GlyRs are likely to be composed of alpha 2 and beta subunits.

Original languageEnglish
Pages (from-to)4353-4363
Number of pages11
JournalJournal of Physiology
Volume592
Issue number19
DOIs
Publication statusPublished - 1 Oct 2014

Keywords

  • AMINOBUTYRIC ACID(A) RECEPTORS
  • RAT CEREBRAL-CORTEX
  • A RECEPTOR
  • POSTNATAL-DEVELOPMENT
  • KINETIC-PROPERTIES
  • MESSENGER-RNAS
  • SUBTYPES
  • EXPRESSION
  • BRAIN
  • ACTIVATION

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