GABA_A receptor agonists modulate intracellular Ca²⁺ levels in activated human CD4⁺ T cells

GABA binding to GABA_A receptors induces transient elevation in intracellular Ca²⁺ concentrations in activated human CD4⁺ T cells. Here we examined the Ca²⁺ response evoked by the GABA_A receptor agonists THIP, muscimol, isoguvacine, and TACA and further, effects of K_V1.3 and KCa3.1 channel blockers on the GABA-evoked Ca²⁺ signal. The cells were activated by anti-CD3 antibody and cultured for 72 h before the effects of agonists and antagonists on intracellular Ca²⁺ levels were examined by Ca²⁺ imaging. mRNA sequencing, qPCR, and immunofluorescence imaging identified the GABA_A subunits expressed. Both the half-maximal concentration for activation (the apparent affinity (EC₅₀)) and the evoked, peak cellular Ca²⁺ response varied among the agonists. The agonists EC₅₀ values varied more than 1,000-fold, but the compounds can, nevertheless, all be classified as high-affinity agonists (EC₅₀ < 1 μM). The EC₅₀ sequence was as follows: GABA (0.005 nM) ≈ THIP (0.005 nM) < muscimol (0.162 nM) < TACA (2.2 nM) < isoguvacine (54.4 nM). In contrast, the peak increase in the cellular Ca²⁺ signal followed the sequence: GABA >> THIP > muscimol > isoguvacine > TACA. For all agonists, the transient Ca²⁺ signal was attenuated at micromolar concentrations. ShK-Dap22 and TRAM-34 that block K_V1.3 and KCa3.1 channels, respectively, inhibited the GABA-activated Ca²⁺ response. The CRAC channel inhibitor YM58483 reduced the Ca²⁺ current by 40%. The pharmacology is consistent with the ρ2 GABA_A receptor subunit being a part of the high-affinity GABA_A receptors expressed in human CD4⁺ T cells. The agonists at the ρ2-containing GABA_A receptors induced intracellular Ca²⁺ signals in human CD4⁺ T cells that were related to the agonist concentration. All tested agonists have a high apparent affinity but differ in efficacy at the receptors. Ca²⁺ signaling enables high-throughput assays for fast screening of potential drugs aimed at GABA_A receptors regulating the T cell functions.