Modulation Of The Glycine Receptor-Mediated Current In Cultured Neurons Of Rat Inferior Colliculus By Arachidonic Acid

BACKGROUND: Arachidonic acid (AA), a polyunsaturated fatty acid with four double bonds, is released from the endoplasmic reticulum and nuclear membrane by the translocation of type IV cytosolic phospholipase A₂ (PLA₂), phospholipase C (PLC), and phospholipase D (PLD). AA has important modulatory effects in the nervous systems and its concentration is altered during some nervous system diseases and aging. In the central nervous systems, AA and its metabolites can modulate almost all ion channels and neurotransmitter transporters. In general, AA exerts an excitory effect on neurons.The glycine receptor (GlyR) is one of essential inhibitory neurotransmitter receptors and is the primary inhibitory receptor in the brainstem and other lower central nervous systems. More and more attention has been focused on the roles of GlyRs in physiological and pathological processes, espacially in development. Currently, the effects of AA on GlyRs are not clear. The purpose of the present study was to understand how AA functionally mediates GlyRs.METHODS: In the present study, cultured IC neurons and HEK293T cells with various subunits of GlyRs expressed were used for examing the effect of AA on the glycine-induced current (I_Gly) using traditional whole-cell patch–clamp recording technique. The membrane potential was held at -60 mV in cultured neurons and -50 mV in HEK293T cells, respectively. Drugs were applied with a rapid application technique called'Y-tube'method.RESULTS: (1) AA inhibited the I_Gly in a concentration-dependent manner. In my experiment, AA (100μM) alone did not induce any detectable current; however, when it was co-applied with glycine, AA concentration-dependently reduced the I_Gly. The IC₅₀ for inhibition of the I_Gly by AA was 24.0±2.3μM. (2) AA produced a rightward shift of the concentration–response curve of the I_Gly and reduced the I_Gly at a saturating concentration. (3) The inhibitory effect of AA on the I_Gly was membrane potential-dependent and AA did not change the ion selectivity of GlyRs. However, the inhibition of the I_Gly by AA was voltage-dependent, being greater at positive membrane potentials than at negative potentials. (4) The inhibition of AA on the I_Gly depended on drug application mode. AA depressed the I_Gly with the a co-application protocol, indicating that the effects of AA on the I_Gly were very fast. With a sequential application protocol, AA significantly reduced the I_Gly, demonstrating that this inhibition did not depend on the opening state of GlyRs. (5) Intracellular AA dialysis had no effect on the inhibition of the I_Gly by AA; however, extracellular administration of AA significantly reduced the I_Gly after intracellular dialysis with AA, indicating that the inhibition of the I_Gly by AA comes from AA that binds to an extracellular site. (6) AA accelerated the desensitization of GlyRs. With the co-application of AA, the desensitization of the I_Gly was accelerated and the desensitization time of the current induced by a saturating concentration of glycine was significantly decreased. (7) The inhibition of AA on GlyRs was subunit-dependent, though it was not subunit-specific. In HEK293T cells expressing homomericα1-,α2-,α3- and heteromericα1β-,α2β-,α3β-GlyRs, AA had significantly weaker inhibitory effects onα1-containing GlyRs than onα2- andα3-containing GlyRs.CONCLUSIONS: My results show that AA could directly reduce the I_Gly in a noncompetitive and subunit-dependent manner, suggesting that AA may modulate the glycinergic transmission and plays an important role in the central nerveous systems. AA most likely serves as an allosteric modulator of GlyRs. My study may help to understand the receptor basis for effects of AA in the central nervous systems.