title: Ionotropic GABA and glycine receptor subunit composition in human pluripotent stem cell-derived excitatory cortical neurones.
abstract: We have assessed, using whole-cell patch-clamp recording and RNA-sequencing (RNA-seq), the properties and composition of GABAA receptors (GABAARs) 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 μm and 182 μm, respectively, and the presence of a GABAAR population displaying low agonist potencies is supported by strong RNA-seq signals for α2 and α3 subunits. GABAAR-mediated currents, evoked by EC50 concentrations of GABA, were blocked by bicuculline and picrotoxin with IC50 values of 2.7 and 5.1 μm, respectively. hECN GABAARs are predominantly γ subunit-containing as assessed by the sensitivity of GABA-evoked currents to diazepam and insensitivity to Zn(2+), together with the weak direct agonist action of gaboxadol; RNA-seq indicated a predominant expression of the γ2 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 β2 or β3 subunit, with RNA-seq analysis indicating strong expression of β3 in hECN GABAARs. Taken together our data support the notion that hECN GABAARs have an α2/3β3γ2 subunit composition - a composition that also predominates in immature rodent cortex. GlyRs expressed by hECNs were activated by glycine with an EC50 of 167 μm. Glycine-evoked (500 μm) currents were blocked by strychnine (IC50 = 630 nm) and picrotoxin (IC50 = 197 μm), where the latter is suggestive of a population of heteromeric receptors. RNA-seq indicates GlyRs are likely to be composed of α2 and β subunits.
authors.affiliation: Centre for Integrative Physiology, University of Edinburgh, Edinburgh, EH8 9XD, UK Centre for Brain Development and Repair, Institute for Stem Cell Biology and Regenerative Medicine, National Centre for Biological Sciences, Bangalore, 560065, India Euan MacDonald Centre for MND Research, University of Edinburgh, Edinburgh, EH16 4SB, UK.
authors.forename: Owain T
authors.initials: OT
authors.lastname: James
authors.affiliation: Centre for Integrative Physiology, University of Edinburgh, Edinburgh, EH8 9XD, UK Euan MacDonald Centre for MND Research, University of Edinburgh, Edinburgh, EH16 4SB, UK.
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Livesey
authors.affiliation: Centre for Integrative Physiology, University of Edinburgh, Edinburgh, EH8 9XD, UK.
authors.forename: Jing
authors.initials: J
authors.lastname: Qiu
authors.affiliation: Centre for Integrative Physiology, University of Edinburgh, Edinburgh, EH8 9XD, UK Centre for Brain Development and Repair, Institute for Stem Cell Biology and Regenerative Medicine, National Centre for Biological Sciences, Bangalore, 560065, India.
authors.forename: Owen
authors.initials: O
authors.lastname: Dando
authors.affiliation: Euan MacDonald Centre for MND Research, University of Edinburgh, Edinburgh, EH16 4SB, UK Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh, EH16 4SB, UK MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, EH16 4SB, UK.
authors.forename: Bilada
authors.initials: B
authors.lastname: Bilican
authors.affiliation: Centre for Integrative Physiology, University of Edinburgh, Edinburgh, EH8 9XD, UK Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh, EH16 4SB, UK.
authors.forename: Ghazal
authors.initials: G
authors.lastname: Haghi
authors.affiliation: Centre for Integrative Physiology, University of Edinburgh, Edinburgh, EH8 9XD, UK Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh, EH16 4SB, UK.
authors.forename: Rinku
authors.initials: R
authors.lastname: Rajan
authors.affiliation: Euan MacDonald Centre for MND Research, University of Edinburgh, Edinburgh, EH16 4SB, UK Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh, EH16 4SB, UK MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, EH16 4SB, UK.
authors.forename: Karen
authors.initials: K
authors.lastname: Burr
authors.affiliation: Centre for Integrative Physiology, University of Edinburgh, Edinburgh, EH8 9XD, UK Patrick Wild Centre, University of Edinburgh, Edinburgh, EH8 9XD, UK.
authors.forename: Giles E
authors.initials: GE
authors.lastname: Hardingham
authors.affiliation: Centre for Brain Development and Repair, Institute for Stem Cell Biology and Regenerative Medicine, National Centre for Biological Sciences, Bangalore, 560065, India Euan MacDonald Centre for MND Research, University of Edinburgh, Edinburgh, EH16 4SB, UK Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh, EH16 4SB, UK MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, EH16 4SB, UK.
authors.forename: Siddharthan
authors.initials: S
authors.lastname: Chandran
authors.affiliation: Centre for Integrative Physiology, University of Edinburgh, Edinburgh, EH8 9XD, UK Centre for Brain Development and Repair, Institute for Stem Cell Biology and Regenerative Medicine, National Centre for Biological Sciences, Bangalore, 560065, India Patrick Wild Centre, University of Edinburgh, Edinburgh, EH8 9XD, UK.
authors.forename: Peter C
authors.initials: PC
authors.lastname: Kind
authors.affiliation: Centre for Integrative Physiology, University of Edinburgh, Edinburgh, EH8 9XD, UK Patrick Wild Centre, University of Edinburgh, Edinburgh, EH8 9XD, UK dwyllie1@staffmail.ed.ac.uk.
authors.forename: David J A
authors.initials: DJ
authors.lastname: Wyllie
mesh_terms: D002540:Cerebral Cortex / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D009118:Muscimol / Q000494:pharmacology; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D021122:Protein Subunits / Q000378:metabolism*; D011963:Receptors, GABA-A / Q000378:metabolism*; D018009:Receptors, Glycine / Q000378:metabolism*; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
chemical_list: D021122:Protein Subunits; D011963:Receptors, GABA-A; D018009:Receptors, Glycine; D009118:Muscimol; D005680:gamma-Aminobutyric Acid