γ1-Containing GABA-A Receptors Cluster at Synapses Where they Mediate Slower Synaptic Currents than γ2-Containing GABA-A Receptors

Abstract

GABA-A receptors (GABAARs) are pentameric ligand-gated ion channels that are assembled mainly from α (α1–6), β (β1–3) and γ (γ1–3) subunits. Although GABAARs containing γ2L subunits mediate most of the inhibitory neurotransmission in the brain, significant expression of γ1 subunits is seen in the amygdala, pallidum and substantia nigra. However, the location and function of γ1-containing GABAARs in these regions remains unclear. In “artificial” synapses, where the subunit composition of postsynaptic receptors is specifically controlled, γ1 incorporation slows the synaptic current decay rate without affecting channel deactivation, suggesting that γ1-containing receptors are not clustered and therefore activated by diffuse neurotransmitter. However, we show that γ1-containing receptors are localized at neuronal synapses and form clusters in both synaptic and extrasynaptic regions. In addition, they exhibit rapid membrane diffusion and a higher frequency of exchange between synaptic and perisynaptic populations compared to γ2L-containing GABAARs. A point mutation in the large intracellular domain and a pharmacological analysis reveal that when a single non-conserved γ2L residue is mutated to its γ1 counterpart (T349L), the synaptic current decay is slowed from γ2L- to γ1-like without changing the clustering or diffusion properties of the receptors. In addition, previous fast perfusion and single channel kinetic experiments revealed no difference in the intrinsic closing rates of γ2L- and γ1-containing receptors when expressed in HEK293 cells. These observations together with Monte Carlo simulations of synaptic function confirm that decreased clustering does not control γ1-containing GABAAR kinetics. Rather, they suggest that γ1- and γ2L-containing receptors exhibit differential synaptic current decay rates due to differential gating dynamics when localized at the synapse.

Physiological and pharmacological properties of inhibitory postsynaptic currents mediated by α5β1γ2, α5β2γ2 and α5β3γ2 GABA A receptors

“…4C). This fits well with our recent Monte Carlo simulations that demonstrated GABAergic IPSC decay time constants are not affected by changes in a variety of synapse parameters, including GABA diffusion coefficient, synaptic cleft width, postsynaptic density size or GABA A R clustering density (Dixon et al, 2017).…”

“…To determine whether the β subunit-dependent variation in IPSC kinetics was due to variations in the intrinsic receptor gating properties or to differential interactions with synapsespecific molecules (Barberis et al, 2011;Dixon et al, 2017), we recorded ensemble currents from outside-out patches excised from HEK293 cells that expressed either α5β1γ2L, α5β2γ2L or α5β3γ2L GABA A Rs. To mimic synaptic activation conditions, we applied a saturating 5 mMGABA concentration for 1 ms via a piezoelectric translation device.…”

“…Moreover, cytoplasmic proteins such as radixin or AP2 clathrin adaptor protein that trap α5β3γ2L GABA A Rs in extrasynaptic compartments (Hausrat et al, 2015;Vien et al, 2015) may not be functional in HEK293 cells. Irrespective of these potential limitations, a range of recombinant GABA A R isoforms has been found to mediate heterosynaptic IPSCs with kinetics similar to those mediated by the same isoforms in neurons (Dixon et al, 2014;Dixon et al, 2015;Dixon et al, 2017;Fuchs et al, 2013;Wu et al, 2012). Because the kinetics of IPSCs mediated by defined α5-containing GABA A Rs in vivo are not known, this comparison is not possible.…”

Inhibitory synapse deficits caused by familial α1 GABAA receptor mutations in epilepsy

“…Atto-647N at the membrane surface. Identification of inhibitory synapses was conducted as described in (35). Briefly, synaptic contacts in neuronal cultures were defined as points of colocalisation between the green puncta found in neurons transfected with tagged wild-type (WT) or mutant 1 subunits and magenta synaptotagmin rich presynaptic contacts of surrounding neurons.…”

Synaptic GABAergic transmission in the central amygdala (CeA) of rats depends on slice preparation and recording conditions

“…Recording temperature affects the excitability of neurons in vitro , with greater activity in cells maintained near physiological temperatures (e.g., Cao and Oertel, ; Lee et al, ; Micheva and Smith, ; Graham et al, ; Baginskas et al, ), and was therefore included as an additional variable in our experiments. Similarly, recording temperature affects neurotransmitter release and subsequent alterations in current flow across the membrane, with increased temperature leading to increased neurotransmitter release and/or faster sIPSC/EPSC kinetics (Otis and Mody, ; Postlehwaite et al, ; Dixon et al, ). Together, our data demonstrate that slicing solution and recording temperature each altered (1) GABAergic transmission onto CeA neurons and (2) the effect of bath‐applied CRF on GABAergic transmission over time, which was further dependent on whether changes of sIPSC properties were reported as raw or normalized measures.…”