Glycine receptors (GlyRs) belong to the family of ligand-gated ion channels which includes receptors for gamma-aminobutyric acid (GABA), serotonin and acetylcholine. GlyRs provide inhibitory neurotransmission mainly in spinal cord and brainstem synapses of vertebrates. They are implicated in the coordination of reflex responses, processing of sensory signals and pain sensation. Dysfunction of these receptors results in hypertonic motor disorders. One of them, hyperkplexia (or startle disease) is a genetic neurological disorder of humans. To struggle with these disorders it is important to develop strategies for increasing an activatory capability of GlyR channels. Function of GlyRs is known to be regulated by protein phosphorylation and several other pathways (Betz et al. 1999). We cloned two GlyR subunits from zebrafish and determined their functional properties. Fusing of the inhibitory GlyR with green fluorescent protein (GFP) allowed us visualization and analysis of distribution of this protein in living cells.

Recently we discovered a novel mechanism of GlyR modulation: rapid potentiation by intracellular Ca²⁺ (Fucile et al. 2000). Using a patch-clamp and imaging techniques we demonstrated that in spinal cord neurons and in the HEK cells expressing homomeric GlyRs: (i) Ca²⁺ influx through receptor-operated or voltage-gated Ca²⁺-permeable channels causes rapid and transient augmentation the amplitude of GlyR currents; (ii) the minimal interval necessary for GlyR channel potentiation is less than 100 ms; (iii) phosphorylation and G-protein pathways do not underlie this phenomenon; (iv) elevation of intracellular Ca²⁺ results in prolongation of single channel burst kinetics; (v) Ca²⁺ potentiates GlyR by increasing its apparent affinity to glycine; (vi) in inside-out patches, exposure of the cytoplasmic side of the membrane to Ca²⁺ had no effect on activity of GlyR channels, suggesting involvement of diffusible factor, presumably a Ca²⁺-binding protein.

To identify proteins interacting with GlyR, we used the cytoplasmic loop of human α1 subunit (GlyRh1) as a bait for two-hybrid screening of a human brain cDNA library. This approach allowed us to identify five new interactors which were then individually co-expressed with GlyRh1 in human cell lines (HEK-293 and CHO cells). Analysis of the concentration dependencies of glycine-induced whole-cell currents revealed that overexpression of one of these proteins results in a 3-5-fold decrease of GlyR sensitivity to agonist.

Our results suggests that Ca²⁺ ions trigger a powerful and rapid modulation of neurotransmission at glycinergic synapses controlling a gating of GlyR channels through a diffusible Ca²⁺-sensitive cytoplasmic intermediate.