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Brain Sci
2021 Mar 30;114:. doi: 10.3390/brainsci11040441.
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Dissecting the Molecular Determinants of GABAA Receptors Current Rundown, a Hallmark of Refractory Human Epilepsy.
Cifelli P, Di Angelantonio S, Alfano V, Morano A, De Felice E, Aronica E, Ruffolo G, Palma E.
???displayArticle.abstract??? GABAA receptors-(Rs) are fundamental for the maintenance of an efficient inhibitory function in the central nervous system (CNS). Their dysfunction is associated with a wide range of CNS disorders, many of which characterized by seizures and epilepsy. Recently, an increased use-dependent desensitization due to a repetitive GABA stimulation (GABAA current rundown) of GABAARs has been associated with drug-resistant temporal lobe epilepsy (TLE). Here, we aimed to investigate the molecular determinants of GABAA current rundown with two different heterologous expression systems (Xenopus oocytes and human embryonic kidney cells; HEK) which allowed us to manipulate receptor stoichiometry and to study the GABAA current rundown on different GABAAR configurations. To this purpose, we performed electrophysiology experiments using two-electrode voltage clamp in oocytes and confirming part of our results in HEK. We found that different degrees of GABAA current rundown can be associated with the expression of different GABAAR β-subunits reaching the maximum current decrease when functional α1β2 receptors are expressed. Furthermore, the blockade of phosphatases can prevent the current rundown observed in α1β2 GABAARs. Since GABAAR represents one important therapeutic target in the treatment of human epilepsy, our results could open new perspectives on the therapeutic management of drug-resistant patients showing a GABAergic impairment.
Figure 1. GABA current rundown in oocytes expressing different GABAAR subunit combinations. (A) Time course of GABA current rundown relative to α1β2γ2(●), α1β1(●), α1β2(●), α1β3(○) GABAARs. GABA currents are normalized to that elicited by the first GABA application (IGABA: ●, 2720 ± 280 nA, n = 24; ●, 1264 ± 106 nA, n = 25; ●, 749±130 nA, n = 95; ○, 875 ± 139 nA, n = 30). (B) Representative evoked GABA currents at first, sixth GABA application of the rundown protocol and relative washout (black bars) (C) The bars represent mean ± SEM of GABA current rundown values in groups of oocytes expressing different GABA receptor subunit combinations as indicated. GABA current (%) has been calculated as the reduction in peak amplitude of the 6th GABA-evoked current as percent of the 1st current after the rundown protocol (six 10-s applications of 500 μM GABA at 40-s intervals). Statistical significance among the four groups was assessed with Kruskal-Wallis One Way Analysis of Variance on ranks with pairwise multiple comparison procedures (Dunn’s Method). * p < 0.05.
Figure 2. GABA current rundown in HEK cells transfected with GABAARs cDNAs. Time course of averaged responses (±SEM; p < 0.05, Mann–Whitney rank sum test) obtained in HEK cells expressing either α1β2γ2 (n = 11, ●) or α1β2 (n = 13, ●) GABAARs. In each cell, peak amplitude of subsequent responses was expressed as a percentage of the first response of the rundown protocol.
Figure 3. Effect of okadaic acid on GABA current rundown in oocytes expressing α1β2 GABAARs. The bars represent mean ± SEM of GABA current rundown values before (red bar) and after (grey bar) the intracellular injection of okadaic acid (≈20 nM final concentration) n = 11; * p < 0.001, Wilcoxon Signed Rank Test. Inset: representative currents in one oocyte at first and sixth GABA application of rundown protocol and relative recovery after application of okadaic acid.
Figure 4. GABAARs affinity in oocytes expressing α1β2γ2 GABAARs or α1β2 GABAARs. GABA dose–current relationships in oocytes injected with α1β2γ2 (●) or α1β2 cDNAs (●). Peak currents were normalized to the current obtained with 1 mM GABA and refer to 6 oocytes each point. EC50 and nH were 7.36 ± 0.47 μM and 1.0 ± 0.04 for α1β2 receptors; 5.0 ± 0.15 μM and 0.99 ± 0.1 for α1β2γ2; p > 0.05, Mann–Whitney rank sum test.
Figure 1. GABA current rundown in oocytes expressing different GABAAR subunit combinations. (A) Time course of GABA current rundown relative to α1β2γ2(●), α1β1(●), α1β2(●), α1β3(○) GABAARs. GABA currents are normalized to that elicited by the first GABA application (IGABA: ●, 2720 ± 280 nA, n = 24; ●, 1264 ± 106 nA, n = 25; ●, 749±130 nA, n = 95; ○, 875 ± 139 nA, n = 30). (B) Representative evoked GABA currents at first, sixth GABA application of the rundown protocol and relative washout (black bars) (C) The bars represent mean ± SEM of GABA current rundown values in groups of oocytes expressing different GABA receptor subunit combinations as indicated. GABA current (%) has been calculated as the reduction in peak amplitude of the 6th GABA-evoked current as percent of the 1st current after the rundown protocol (six 10-s applications of 500 μM GABA at 40-s intervals). Statistical significance among the four groups was assessed with Kruskal-Wallis One Way Analysis of Variance on ranks with pairwise multiple comparison procedures (Dunn’s Method). * p < 0.05.
Figure 2. GABA current rundown in HEK cells transfected with GABAARs cDNAs. Time course of averaged responses (±SEM; p < 0.05, Mann–Whitney rank sum test) obtained in HEK cells expressing either α1β2γ2 (n = 11, ●) or α1β2 (n = 13, ●) GABAARs. In each cell, peak amplitude of subsequent responses was expressed as a percentage of the first response of the rundown protocol.
Figure 3. Effect of okadaic acid on GABA current rundown in oocytes expressing α1β2 GABAARs. The bars represent mean ± SEM of GABA current rundown values before (red bar) and after (grey bar) the intracellular injection of okadaic acid (≈20 nM final concentration) n = 11; * p < 0.001, Wilcoxon Signed Rank Test. Inset: representative currents in one oocyte at first and sixth GABA application of rundown protocol and relative recovery after application of okadaic acid.
Figure 4. GABAARs affinity in oocytes expressing α1β2γ2 GABAARs or α1β2 GABAARs. GABA dose–current relationships in oocytes injected with α1β2γ2 (●) or α1β2 cDNAs (●). Peak currents were normalized to the current obtained with 1 mM GABA and refer to 6 oocytes each point. EC50 and nH were 7.36 ± 0.47 μM and 1.0 ± 0.04 for α1β2 receptors; 5.0 ± 0.15 μM and 0.99 ± 0.1 for α1β2γ2; p > 0.05, Mann–Whitney rank sum test.
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