Protein Structure
The first primary sequence of a reuptake protein was published in 1990. The technique for protein sequence determination relied upon the purification, sequencing, and cloning of the transporter protein in question, or expression cloning strategies in which transport function was used as an assay for cDNA species coding for that transporter. After separate investigations had sequenced the DNA that coded for both GABA transporter and norepinephrine transporter, it was realized that there were many similarities between the two DNA sequences. Further exploration in the field of reuptake proteins found that many of the transporters associated with important neurotransmitters within the body were also very similar in sequence to the GABA and norepinephrine transporters. The members of this new family include transporters for dopamine, norepinephrine, serotonin, glycine, proline and GABA. They were called Na+/Cl- dependent neurotransmitter transporters. Sodium and Chloride ion dependence will be discussed later in the mechanism of action. Using the commonalities among sequences and hydropathy plot analyses, it was predicted that there are 12 hydrophobic membrane spanning regions in the ‘Classical’ transporter family. In addition to this, the N- and C-termini exist in the intracellular space. These proteins also all have an extended extracellular loop between the third and fourth transmembrane sequences. Site-directed chemical labeling experiments verified the predicted topological organization of the serotonin transporter.
In addition to neurotransmitter transporters, many other proteins in both animals and prokaryotes were found with similar sequences, indicating a larger family of Neurotransmitter:Sodium Symporters (NSS). One of these proteins, LeuT, from Aquifex aeolicus, was crystallized by Yamashita et al. with very high resolution, revealing a molecule of leucine and two Na+ ions bound near the center of the protein. They found that the transmembrane (TM) helices 1 and 6 contained unwound segments in the middle of the membrane. Along with these two helices, TM helices 3 and 8 and the areas surrounding the unwound sections of 1 and 6 formed the substrate and sodium ion binding sites. The crystal structure revealed pseudo-symmetry in LeuT, in which the structure of TM helices 1-5 is reflected in the structure of helices 6-10.
There is an extracellular cavity in the protein, into which protrudes a helical hairpin formed by extracellular loop EL4. In TM1, an aspartate distinguishes monoamine NSS transporters from amino acid transporters which contain a glycine at the same position. External and internal “gates” were assigned to pairs of negatively and positively charged residues in the extracellular cavity and near the cytoplasmic ends of TM helices 1 and 8.
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