Friedman CA, Russell BJ, Kohls MD, Ancheta LR, Shramm PA, Lappi DA (2016) Targeting vesicular gaba transporter (vGAT)-expressing cells with a polyclonal antibody to the lumenal domain of vGAT: results with a saporin conjugate. Neuroscience 2016 Abstracts 124.06 / E30. Society for Neuroscience, San Diego, CA. PMID: 0
Summary: The vesicular GABA transporter (vGAT) mediates the accumulation of GABA into synaptic vesicles and the release from these vesicles. vGAT is expressed in nerve endings of GABAergic neurons throughout the CNS. The GABAergic system is crucial for the development and functional maturation of the nervous system, as well as the maintenance of balance between excitation and inhibition required for normal neural circuit function. A panel of research tools has been created that target the lumenal domain of vGAT. Antiserum was raised against a peptide from the C-terminus of rat vGAT and resulted in an affinity-purified antibody and an immunotoxin specific for vGAT-expressing cells. The antigen sequence is identical among human, rat, mouse, pig and guinea pig. A stably-transfected clone of HEK293 cells (2E11HEK) that expresses vGAT on the cell surface shows excellent results for western blot, ICC and flow cytometry using both the antiserum and affinity-purified antibody. The affinity-purified antibody was used to create an immunotoxin by conjugating it to the ribosome-inactivating protein, saporin. Saporin irreversibly inactivates ribosomes, blocking protein synthesis, when it is escorted into a cell. Saporin cannot enter a cell on its own, but when escorted by something that binds to a cell surface marker it is internalized along with the binding moiety and causes cell death. The immunotoxin (Anti-vGAT-SAP) is 1000-fold more cytotoxic to 2E11HEK cells than non-conjugated saporin, based on the EC50 in a cytotoxicity assay. The affinity-purified vGAT antibody binds specifically to cells that express vGAT, and delivers a payload to the interior of these cells. Anti-vGAT-SAP could be an important tool in studying diseases involving dysfunction of GABAergic neurons. GABAergic neuron dysfunction is thought to be an underlying factor in Epilepsy, Down Syndrome, Fragile X Syndrome, Schizophrenia and Autism. In vivo, elimination of vGAT-expressing cells in a particular area (rather than knocking out vGAT systemically) makes it possible to study the functions of those regional cells. Animals can then be tested behaviorally before and after injections of Anti-vGAT-SAP to demonstrate the effects of loss of cells in a particular region of interest.