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

GABA binding to GABAA receptors induces transient elevation in intracellular Ca2+ concentrations in activated human CD4+ T cells. Here we examined the Ca2+ response evoked by the GABAA receptor agonists THIP, muscimol, isoguvacine, and TACA and further, effects of KV1.3 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 GABAA 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 KV1.3 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 GABAA receptor subunit being a part of the high-affinity GABAA receptors expressed in human CD4+ T cells. The agonists at the ρ2-containing GABAA 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 GABAA receptors regulating the T cell functions.