Glycine acts as an inhibitory neurotransmitter in many interneurons of the spinal cord, brain stem and other regions of the central nervous system by activating the strychnine-sensitive inhibitory glycine receptor. In addition, it is an essential coagonist of the NMDA subtype of excitatory glutamate receptors. At both receptors, the concentration of glycine in the synaptic cleft must be precisely regulated. This is accomplished by two high affinity transporters located in the plasma membranes of neuronal (GlyT2) and predominantly glial (GlyT1) and cells. Both are members of the Slc6a superfamily of Na+/Cl- dependent neurotransmitter transporters share a common membrane topology including 12 membrane spanning regions.
Our group pursues different strategies to investigate the molecular and biochemical properties and physiological function of these transporters.
Based on structural models, generated on the basis of the crystal structure of the bacterial ortholog LeuTA we try to identify residues important for transporter function by mutagenesis and recombinant expression in a variety of cells. Transporter function is analyzed by biochemical as well as electrophysiological methods. Furthermore we try to identify new interacting proteins by the application of the yeast 2-hybrid system.
In an additional approach we probe the in-vivo function of glycine transporters in mice displaying altered GlyT expression. Mutant mice were analyzed by biochemical, immunocytochemical, electrophysiological and behavioural methods.
To unravel the in-vivo functions of both GlyT1 and GlyT2 we used conventional gene targeting to generate mouse lines lacking expression of the respective transporters. The first analysis of the different glycine transporter mutant mice discloses distinct but essential roles for the two glycine transporters in the early postnatal life.
GlyT1 deficient mice die within the first 12 h after birth, showing a strong hypotonic phenotype accompanied by respiratory depression and an inability to suckle mimicking the symptoms of the human disease non-ketotic hyperglycenemia (NKH). Biochemical and immunhistochemical data failed to reveal any morphological differences between wildtype and mutant mice. Electrophysiological recordings from hypoglossal motoneurons of GlyT1-/- mice revealed a strong activation of strychnine sensitive glycine receptors caused by accumulation of glycine in the synaptic cleft. Taken together these data prove that in the neonatal animal GlyT1 plays an essential role for the regulation of glycine concentrations in the cerebrospinal fluid in brainstem and spinal cord.
In contrast, GlyT2 knockout mice are normal at birth but develop a complex neuromotor phenotype including spastic seizures, spontaneous tremors, and an impaired righting response leading subsequently to their death during the third postnatal week. Extensive biochemical and immunhistological analysis failed to reveal abnormalities in mutant animals. Electrophysiological studies demonstrate reduced amplitudes in mIPSC. Thus, GlyT2 is essential for the efficient reuptake of glycine into the glycinergic presynaptic terminal and thereby providing the basis to maintain high glycine levels in synaptic vesicles.
The phenotype seen in these animals resembles most of the symptoms seen in human hyperekplexia patients, a rare hereditary disease, that was previously associated with mutations in the Glycine receptor genes. Indeed, sequencing of the GlyT2 gene of patients that were diagnosed for hyperekplexia but have been found to be negative for mutations in other hyperplexia associated genes revealed several mutations in the GlyT2 gene. In part these mutations within the GlyT2 coding sequences resulted in a dysfunctional transporter. Together these data provide evidence that the GlyT2 gene is indeed a candidate gene for human hyperekplexia.
Another focus of our work is how these transporters are assembled and inserted into the lipid bilayer? This includes the questions which structural elements determine the sorting and subcellular localization of these proteins in the plasma membrane?
In previous studies using the yeast two hybrid system our group identified syntenin I, a PDZ domain containing protein as an interaction partner of the extreme C-terminus of GlyT2. Other putative interacting proteins are currently analyzed.
The function of the PDZ domain ligand motif of GlyT2 was subsequently analyzed by a loss of function approach. Mutant transporters were normally inserted into the plasma membrane and did not display impaired transport activity as analyzed in HEK293 cells. Upon expression in hippocampal neurons, however, inactivation of the PDz ligand motif resulted in a reduction in synaptic localization of the transporter,
These results were consistent with the idea, that the PDZ ligand motif is important for stabilizing GlyT2 at synaptic sites.
Due to their role in regulation of synaptic glycine levels, GlyT1 and GlyT2 have been shown to be involved in the control of fundamental physiological processes including motor function, pain, learning and memory. Therefore, the analysis of the function of GlyTs on a molecular level as well as their function in-vivo and might reveal insights in the modulation of neurotransmission by neurotransmittter transporters and possibly new strategies for the treatment of human diseases.