Premium
Ionotropic GABA and glycine receptor subunit composition in human pluripotent stem cell‐derived excitatory cortical neurones
Author(s) -
James Owain T.,
Livesey Matthew R.,
Qiu Jing,
Dando Owen,
Bilican Bilada,
Haghi Ghazal,
Rajan Rinku,
Burr Karen,
Hardingham Giles E.,
Chandran Siddharthan,
Kind Peter C.,
Wyllie David J. A.
Publication year - 2014
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jphysiol.2014.278994
Subject(s) - ionotropic effect , excitatory postsynaptic potential , neuroscience , induced pluripotent stem cell , glycine receptor , chemistry , glycine , gabaa receptor , protein subunit , biology , receptor , nmda receptor , inhibitory postsynaptic potential , biochemistry , embryonic stem cell , amino acid , gene
Key points This study reports a functional assessment of the subunit composition of inhibitory ionotropic GABA A receptors (GABA A Rs) and glycine receptors (GlyRs) expressed by excitatory cortical neurones derived from human embryonic stem cells (hECNs). GABA A Rs expressed by hECNs are predominantly composed of α2/3β3γ2 subunits; such a composition is typical of that reported for GABA A Rs expressed in rodent embryonic cortex. Analysis of GlyRs expressed by hECNs indicates they are likely to contain α2 and β subunits – a composition in rodents that is associated with a late embryonic/early postnatal period of development.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 EC 50 values of 278 μ m and 182 μ m , respectively, and the presence of a GABA A R population displaying low agonist potencies is supported by strong RNA‐seq signals for α2 and α3 subunits. GABA A R‐mediated currents, evoked by EC 50 concentrations of GABA, were blocked by bicuculline and picrotoxin with IC 50 values of 2.7 and 5.1 μ m , respectively. hECN GABA A Rs 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 GABA A Rs. Taken together our data support the notion that hECN GABA A Rs 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 EC 50 of 167 μ m . Glycine‐evoked (500 μ m ) currents were blocked by strychnine (IC 50 = 630 n m ) and picrotoxin (IC 50 = 197 μ m ), 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.