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

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.