Abstract
Glycine is an inhibitory neurotransmitter in the spinal cord and brain stem, where it acts on strychnine-sensitive glycine receptors, and is also an excitatory neurotransmitter throughout the brain and spinal cord, where it acts on the N-methyl-d-aspartate family of receptors. There are two Na(+)/Cl(-)-dependent glycine transporters, GLYT1 and GLYT2, which control extracellular glycine concentrations and these transporters show differences in substrate selectivity and blocker sensitivity. A bacterial Na(+)-dependent leucine transporter (LeuT(Aa)) has recently been crystallized and its structure determined. When the amino acid residues within the leucine binding site of LeuT(Aa) are aligned with residues of the two glycine transporters there are a number of identical residues and also some key differences. In this report, we demonstrate that the LeuT(Aa) structure represents a good working model of the Na(+)/Cl(-)-dependent neurotransmitters and that differences in substrate selectivity can be attributed to a single difference of a glycine residue in transmembrane domain 6 of GLYT1 for a serine residue at the corresponding position of GLYT2.
Highlights
The differential expression patterns and physiological roles of the glycine transporter subtypes have recently been exploited in the development of novel transport inhibitors to treat schizophrenia [5,6,7] (GLYT1 inhibitors)
We have focused on the role of the corresponding residues in GLYT1 and GLYT2 in an attempt to explain why GLYT1 can transport the N-methyl derivative of glycine, sarcosine, while the GLYT2 subtype does not bind or transport sarcosine
We have directly tested the suggestion that LeuTAa is a good structural model for the neurotransmitter transporters and used this to better understand differences in substrate selectivity between the two glycine transporter subtypes, GLYT1 and GLYT2
Summary
Chemicals—All chemicals were obtained from Sigma (Sydney, Australia) unless otherwise stated. A rabbit anti-GLYT1 antibody was obtained from David Pow (University of Newcastle, Australia) and used at a dilution of 1: 1000. Charge-to-flux Ratios—Uptake of [3H]glycine (Amersham Biosciences, Sydney, Australia) was measured under voltageclamp in oocytes expressing wild type and mutant transporters. Oocytes were voltage-clamped at Ϫ60 mV and 30 M [3H]glycine was applied for 1 min and the transport current was recorded. This was followed by a 3-min washout and oocytes were removed from the bath and lysed in 50 mM NaOH, and scintillation counting was performed. Data for sarcosine-elicited currents were normalized to the maximal current generated by glycine for each cell
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