Sodium Salicylate Specifically Inhibits The Current Mediated By Glycine Receptors Containing α1-subunits

Aspirin or its metabolite sodium salicylate(NaSal) is perhaps the most widely used drug in the world.It is estimated that 20-30 billion aspirin tablets are consumed annually in the United States alone.Aspirin or NaSal is prescribed for a number of medical purposes ranging from pain relief to stroke prevention due to its wide range of pharmacological actions.Recently,aspirin has been used for prevention and treatment of cardio cerebrovascular diseases for its antiplatelet aggregation effect. Besides gastrointestinal toxicity,aspirin or NaSal at a high dose can cause toxic symptoms such as seizures and tinnitus in the nervous systems.It is reported that the origin of tinnitus is from the central auditory system by the microPET image evidence. This means that NaSal targets central auditory system rather than peripheral auditory system to induce tinnitus in animal models and patients.NaSal can accumulate high concentration in the cerebral spinal fluid of salicylate-induced animal models and patients because it can penetrate the blood-brain-barrier.It is believed that the imbalance between excitation and inhibition is one of the mechanisms underlying the salicylate-induced tinnitus.Previous studies carried out in our lab showed that NaSal could directly inhibite the GABAergic neurotransmission in the auditory cortex and inferial colliculus.Aside from A type GABA receptor(GABA_AR),Glycine receptor(GIyR),which is the member of the superfamily of ligand-gated ion channel/receptors,mediates the fast inhibitory neurotransmission in central nervous system.Although there is no evidence showing that there are functional glycinergic neurotransmission in hippocampal,there is immunity histochemical evidence showing there are heteromericαβGlyRs in the synapse.It has been reported that GlyRs in the hippocampus may be tonically activated and modulate the hyper excitability. Previous studies including our research suggested the tonic activation of GlyRs contributes to the cross-talk between GlyRs and GABA_ARs and the short-term plasticity.The GlyRs is critical for the shaping of the response property of the neurons of central auditory system.Salicylate is recently reported to target a number of neuronal ion channels/receptors;however,the underlying molecular processes remain poorly understood.Here,we report that salicylate's pharmacological action on GlyRs is α1-subunit specific and conferred by a single residue in the first transmembrane segment.With whole-cell patch-clamp recordings,1 mM NaSal was shown to effectively inhibit the maximal current induced by 3 mM glycine in cultured neurons of rat inferior colliculus without significantly altering the EC₅₀ value and the Hill coefficient,suggesting a non-competitive blocking action of salicylate on GlyRs. Intracellular dialysis of NaSal had no effects on the inhibition of glyince-induced current by NaSal,indicating that the acting site is not located in intracellular.In HEK293T cells transfected with either ofα1-,α2-,α3-,α1β-,α2β- orα3β-GlyRs, NaSal only inhibited the current mediated by recombinantα1- orα1β- GlyRs, indicating that salicylate specifically acts on theα1-subunit.NaSal's blocking action was abolished by a single site mutation of isoleucine to valine at position 240 in the first membrane-spanning region of homomericα1-GlyRs.The inhibiton of salicylate on the mutated GlyRs of G254A is same to the wild type GlyRs,which indicating that the isoleucine at position 240 rather than the glycine at the position 254 is important for the inhibiton of salicylate on the GlyRs.In summary,NaSal noncompetitively andα1-subunit specifically depresses glycine-induced currents.This pharmacological action is conferred by the isoleucine residue at position 240 in the first transmembrane segment ofα1-subunit.We suggest that the inhibitory action of NaSal on GlyRs may raise neural excitability and contribute to NaSal-induced toxic symptoms such as seizures and tinnitus.