Enhanced macroscopic desensitization shapes the response of α4 subtype‐containing GABA_A receptors to synaptic and extrasynaptic GABA

Abstract

Up-regulation of the GABA A receptor α4 subunit subtype has been consistently shown in multiple animal models of chronic epilepsy. This isoform is expressed in both thalamus and hippocampus and is likely to play a significant role in regulating corticothalamic and hippocampal rhythms. However, little is known about its physiological properties, thus limiting understanding of the role of α4 subtype-containing GABA A receptors in normal and abnormal physiology. We used rapid GABA application to recombinant GABA A receptors expressed in HEK293T cells to compare the macroscopic kinetic properties of α4β3γ2L receptors to those of the more widely distributed α1β3γ2L receptors. These receptor currents had similar peak current amplitudes and GABA EC 50 values. However, α4β3γ2L currents activated more slowly when exposed to submaximal GABA concentrations, had more fast desensitization (τ = 15-100 ms), and had less residual current during long GABA applications. In addition, α4β3γ2L currents deactivated more slowly than α1β3γ2L currents. Peak currents evoked by repetitive, brief GABA applications were more strongly attenuated for α4β3γ2L currents than α1β3γ2L currents. Moreover, the time required to recover from desensitization was prolonged in α4β3γ2L currents compared to α1β3γ2L currents. We also found that exposure to prolonged low levels of GABA, similar to those that might be present in the extrasynaptic space, greatly suppressed the response of α4β3γ2L currents to higher concentrations of GABA, while α1β3γ2L currents were less affected by exposure to low levels of GABA. Taken together, these data suggest that α4β3γ2L receptors have unique kinetic properties that limit the range of GABA applications to which they can respond maximally. While similar to α1β3γ2L receptors in their ability to respond to brief and low frequency synaptic inputs, α4β3γ2L receptors are less efficacious when exposed to prolonged tonic GABA or during repetitive stimulation, as may occur during learning and seizures. GABA A receptors are pentameric cys-loop receptors composed primarily of two α subunits, two β subunits, and either a δ or a γ subunit selected from six α, three β, one δ, and three γ subunit subtypes. The distribution of specific subtypes is highly brain region and cell type specific, and varies during development and in certain disease states. The presence of a specific subunit subtype confers different pharmacological and physiological properties to receptor isoforms. For example, α subtypes strongly influence GABA A receptor pharmacology. When assembled with β and γ subunits, GABA A receptors containing α1, 2, 3 or 5 subtypes are highly diazepam sensitive. However, α1 subtype-containing receptors are much more sensitive to zolpidem than receptors containing α2 or α3 subtypes, and those containing α5 subtypes are completely insensitive to this drug. In contrast, GABA A receptors containing α4 or α6 subtypes are insensitive to both diazepam and zolpidem. Furthermore, the imidazobenzodiazepine Ro 15-4513, which is an inverse benz...

Effect of the α subunit subtype on the macroscopic kinetic properties of recombinant GABAA receptors

“…α3-containing receptors had the slowest activation rate, while the other receptor isoforms all had activation rates near 1 ms. The slow activation of α3 receptors has been found in previous studies (Gingrich et al, 1995), although our results show faster rates than others have found for α1-and α4-containing receptors (McClellan and Twyman, 1999;Lagrange et al, 2007). The slow activation rate associated with the α3 subtype could also influence our ability to accurately measure the GABA EC 50 and to detect fast components of desensitization for these receptors.…”

“…It is also possible that the relatively slow application rate associated with the whole-cell recording configuration could influence our ability to accurately capture the peak current, and therefore alter the measured EC 50 . In addition, the disruption of interactions of the receptor with cytoskeletal proteins through the process of patch excision could also alter channel properties and could result in differences between whole-cell and outside-out patch recordings (Chen and Olsen, 2007;Lagrange et al, 2007).…”

“…Some of these differences might be due to differences in the subunit composition of the receptors. Previous results from recombinant receptors suggest that the α subtype influences desensitization and deactivation kinetics, although these studies used a variety of experimental protocols and findings are often inconsistent among different laboratories (Gingrich et al, 1995;Tia et al, 1996aTia et al, , 1996bLavoie et al, 1997;Burgard et al, 1999;McClellan and Twyman, 1999;Bianchi et al, 2002a;Lagrange et al, 2007). In order to directly compare the effect of the α subtype on channel properties, we examined the macroscopic kinetic properties of recombinant receptors containing each of the different α subtypes combined with the same β and γ subunit subtypes and under the same recording conditions.…”

Enhancement of GABA A -current run-down in the hippocampus occurs at the first spontaneous seizure in a model of temporal lobe epilepsy

“…This alteration has been hypothesized to be proepileptic because (1) epileptogenic insults (lithium-pilocarpine treatment) increase the α1 subunit expression in immature rats, which are less susceptible than adults to the development spontaneous seizures (28); (2) the α1 subunit is significantly more expressed in slowkindling than in fast kindling rats (29); (3) viral vector-mediated correction of the alteration in the α1/α4 ratio inhibits epilepsy development in the pilocarpine model (30). Interestingly, a recent study (31) shows that α4-containing GABA A channels possess characteristics that imply a reduced GABA response to high frequency stimulation: reduced activation rate, prolonged desensitization, reduced response to repetitive GABA application (i.e., increased run-down). None of these studies, however, describes the evolution of these molecular changes with the natural history of the disease and their correlation with the appearance and maintenance of increased run-down.…”

Glycosylation of β2 Subunits Regulates GABAA Receptor Biogenesis and Channel Gating

“…Recording and Analysis of Macroscopic GABA Evoked Currents-As described before (17) Ϫ equilibrium potential across the cell membrane was close to zero with the combination of external and internal solutions. Cells were voltage-clamped at Ϫ20 mV using an Axopatch 200A amplifier (Axon Instrument).…”