Mechanisms of H⁺ modulation of glycinergic response in rat sacral dorsal commissural neurons

Many ionotropic receptors are modulated by extracellular H⁺. So far, few studies have directly addressed the role of such modulation at synapses. In the present study, we investigated the effects of changes in extracellular pH on glycinergic miniature inhibitory postsynaptic currents (mIPSCs) as well as glycine-evoked currents (I_Gly) in mechanically dissociated spinal neurons with native synaptic boutons preserved. H⁺ modulated both the mIPSCs and I_Gly biphasically, although it activated an amiloride-sensitive inward current by itself. Decreasing extracellular pH reversibly inhibited the amplitude of the mIPSCs and I_Gly, while increasing external pH reversibly potentiated these parameters. Blockade of acid-sensing ion channels (ASICs) with amiloride, the selective antagonist of ASICs, or decreasing intracellular pH did not alter the modulatory effect of H⁺ on either mIPSCs or I_Gly. H⁺ shifted the EC₅₀ of the glycine concentration-response curve from 49.3 ± 5.7 μM at external pH 7.4 to 131.5 ± 8.1 μM at pH 5.5, without altering the Cl^- selectivity of the glycine receptor (GlyR), the Hill coefficient and the maximal I_Gly, suggesting a competitive inhibition of I_Gly by H⁺. Both Zn²⁺ and H⁺ inhibited I_Gly. However, H⁺ induced no further inhibition of I_Gly in the presence of a saturating concentration of Zn²⁺. In addition, H⁺ significantly affected the kinetics of glycinergic mIPSCs and I_Gly. It is proposed that H⁺ and/or Zn²⁺ compete with glycine binding and inhibit the amplitude of glycinergic mIPSCs and I_Gly. Moreover, binding of H⁺ induces a global conformational change in GlyR, which closes the GlyR Cl^- channel and results in the acceleration of the seeming desensitization of I_Gly as well as speeding up the decay time constant of glycinergic mIPSCs. However, the deprotonation rate is faster than the unbinding rate of glycine from the GlyR, leading to reactivation of the undesensitized GlyR after washout of agonist and the appearance of a rebound I_Gly. H⁺ also modulated the glycine cotransmitter, GABA-activated current (I_GABA). Taken together, the results support a 'conformational coupling' model for H⁺ modulation of the GlyR and suggest that H⁺ may act as a novel modulator for inhibitory neurotransmission in the mammalian spinal cord.