Anti-SLC6A1 polyclonal antibody

Objective Sodium-dependent and chloride-dependent gamma-aminobutyric acid (GABA) transporter 1 (SLC6A1) is the target of a number of drugs of clinical importance and is a major determinant of synaptic GABA concentrations. We resequenced the human SLC6A1 gene previously and discovered a novel 21 bp insertion in the predicted promoter region that creates a second tandem copy of the sequence. Here we sought to determine the functional relevance of this variation. Methods We used reporter assays, mobility shift assays, quantitative PCR, and proteomics methods as well as postmortem expression analysis for this work. Results Reporter assays showed that the insertion allele significantly increases promoter activity in multiple cell lines. The zinc finger transcription factor ZNF148 was found to significantly transactivate the promoter and increase expression when overexpressed but could not account for the differences in activity between the two alleles of the promoter. Copy number of the insertion sequence was associated with exponentially increasing activity of a downstream promoter, suggesting that the insertion sequence has enhancer activity when present in multiple copies. SLC6A1 promoter genotype was found to predict SLC6A1 RNA expression in human postmortem hippocampal samples. These results suggest that the insertion polymorphism leads to increased SLC6A1 promoter activity because, in part, of creation of an enhancer element when present as multiple copies. Genotyping individuals from Tanzania in this study suggested that the insertion allele has its origin in Africa. Conclusion On account of the effect of the insertion on promoter activity, this relatively common polymorphism may prove useful in predicting clinical response to pharmacological modulators of SLC6A1 as well as GABAergic function in individuals of African descent. Pharmacogenetics and Genomics 19:53-65 (C) 2009 Wolters Kluwer Health | Lippincott Williams & Wilkins.

Guanidinoacetate (GM) is a biosynthetic precursor of creatine, which plays a critical role in homeostasis of high-energy phosphates in the brain, but cerebral accumulation of GM leads to neurological complications, such as epilepsy and seizures. The purpose of the present study was to clarify the contribution of the gamma-aminobutyric acid (GABA) transport systems to GM transport in astrocytes by means of uptake studies in rat brain slices, primary astrocyte cultures and Chinese hamster ovary (CHO) cells expressing human GABA transporters (GATs). GAA uptake by rat brain slices was Na+- and Cl--dependent, and GABA-sensitive. The inhibitory effect of GABA, a common substrate of GATs, on GM uptake by the brain slices was similar to that of beta-alanine, a selective substrate of GAT2/Slc6a13, GAT3/Slc6a11, and taurine transporter (TauT)/Slc6a6. Taurine, a high-affinity substrate of TauT/Slc6a6, exhibited a lesser inhibitory effect. In contrast, betaine, a substrate of betaine-GABA transporter 1 (BGT1)/Slc6a12, and creatine, a substrate of creatine transporter (CRT)/Slc6a8, had little inhibitory effect. A similar inhibition profile was observed in primary-cultured astrocytes. CHO cells expressing human GAT2/SLC6A13, GAT3/SLC6A11 and BGT1/SLC6Al2 exhibited GM transport, whereas CHO cells expressing GAT1/SLC6A1 did not. The Michaelis-Menten values in CHO cells expressing GAT2/SLC6A13 and GAT3/SLC6A11 were similar to those in primary-cultured astrocytes. Overall, our results suggest that astrocytic GAT2/Slc6a13 and GAT3/Slc6a11 play major roles in GM uptake as regulatory mechanisms of GM in rat brain, while TauT/Slc6a6, BGT1/Slc6a12, and CRT/Slc6a8 make relatively small contributions. (C) 2017 Elsevier Ltd. All rights reserved.