Ca-DEPENDENT MODULATION OF HUMAN GLYCINE RECEPTORS EXPRESSED IN CULTURED CELL LINES
S. Buldakova,1 E. Real,2 Y. Jacob,2 P. Bregestovski 1
1 Institut de Neurobiologie de la Méediterranée (INMED), 13273 Marseille, and
2 Institut Pasteur, 25 Rue du Dr. Roux, 75724 Paris, France;
1 e-mail: pbreges@inmed.univ-mrs.fr
Glycine receptors (GlyRs) provide the main inhibitory neurotransmission in spinal cord and brainstem synapses of
vertebrates. Fucile et al. (2000) discovered that elevation of intracellular Ca2+ caused rapid
potentiation of GlyRs. This modulation develops in less than 100 ms. It is characterized by an increase in GlyR
apparent affinity for glycine. It has been suggested that the phenomenon of Ca-induced potentiation involves an
unknown Ca2+-binding protein (CaBP). Using the yeast two-hybrid system, screening of human brain cDNA
library against the cytoplasmic loop of human alpha 1 subunit (GlyRh1) allowed us to identify five new interactors.
One of them belongs to a family of Ca-binding proteins. We analyzed effect of "short" forms of this protein (CaBP-S)
on functional properties of GlyRh1 expressed in HEK-293 and CHO cells. Using whole-cell recordings and rapid agonist
application we constructed concentration dependencies of glycine-induced currents. This analysis revealed statistical
differences in EC50s between control cells (expressing only GlyRh1) and those expressing CaBP-S. In HEK-293 cells
recorded under conditions of low intracellular Ca concentration (BAPTA 20 mM in the recording pipette), EC50 for
glycine in control cells and expressing GlyRh1 + CaBP-S were, correspondently, 68 ± 49 μM (n = 29) and
409 ± 421 μM (n = 60). In CHO cells EC50 were 54 ± 43 μM (n = 25) and 123 ± 104 μM
(n = 28). These differences were statistically not significant at recording with intracellular solution
containing high Ca concentration (50 μM). In this case EC50 were correspondently 35 ± 28 μM (n = 7)
and 64 ±38 μMM (n = 7). These results suggest that CaBP-S causes decrease of GlyR sensitivity to agonist
through interaction with cytoplasmic domain of GlyR.
Key words: glycine receptor, intracellular calcium, Ca2+-binding protein, cell lines
HEK-293 and CHO, ion currents
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