Zn2+ inhibits glycine transport by glycine transporter subtype 1b.
In the central nervous system, glycine is a co-agonist with glutamate at the N-methyl-D-aspartate subtype of glutamate receptors and also an agonist at inhibitory, strychnine-sensitive glycine receptors. The GLYT1 subtypes of glycine transporters (GLYTs) are responsible for regulation of glycine at excitatory synapses, whereas a combination of GLYT1 and GLYT2 subtypes of glycine transporters are used at inhibitory glycinergic synapses. Zn2+ is stored in synaptic vesicles with glutamate in a number of regions of the brain and is believed to play a role in modulation of excitatory neurotransmission. In this study we have investigated the actions of Zn2+ on the glycine transporters, GLYT1b and GLYT2a, expressed in Xenopus laevis oocytes and we demonstrate that Zn2+ is a noncompetitive inhibitor of GLYT1 but has no effect on GLYT2. We have also investigated the molecular basis for these differences and the relationship between the Zn2+ and proton binding sites on GLYT1. Using site-directed mutagenesis, we identified 2 histidine residues, His-243 in the large second extracellular loop (ECL2) and His-410 in the fourth extracellular loop (ECL4), as two coordinates in the Zn2+ binding site of GLYT1b. In addition, our study suggests that the molecular determinants of proton regulation of GLYT1b are localized to the 2 histidine residues (His-410 and His-421) of ECL4. The ability of Zn2+ and protons to regulate the rate of glycine transport by interacting with residues situated in ECL4 of GLYT1b suggests that this region may influence the substrate translocation mechanism.
Ju
Pengchu
P
Department of Pharmacology, Institute for Biomedical Research, University of Sydney, Sydney, New South Wales 2006, Australia.
Aubrey
Karin R
KR
Vandenberg
Robert J
RJ
D000595:Amino Acid Sequence; D026921:Amino Acid Transport Systems, Neutral / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D001921:Brain / Q000378:metabolism; D050487:Glycine Plasma Membrane Transport Proteins; D006801:Humans; D008969:Molecular Sequence Data; D011522:Protons; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism; D016415:Sequence Alignment; D014981:Xenopus; D015032:Zinc / Q000378:metabolism*
D026921:Amino Acid Transport Systems, Neutral; D050487:Glycine Plasma Membrane Transport Proteins; D011522:Protons; D016194:Receptors, N-Methyl-D-Aspartate; C493271:SLC6A5 protein, human; C493268:SLC6A9 protein, human; D015032:Zinc