IDENTIFICATION AND CHARACTERIZATION OF ANTIDEPRESSANT-SENSITIVE SEROTONIN TRANSPORTER PROTEINS USING SITE-SPECIFIC ANTIBODIES

Serotonin (5HT) transporters (SERTs) are responsible for clearance of synaptic and plasma 5HT and are molecular targets for multiple therape utic and addictive compounds. Recently brain and peripheral SERT cDNAs have been cloned and characterized functionally in transfected cells. Antipeptide (S365) and anti-fusion protein (CT-2) antibodies, directe d at epitopes poorly conserved among other Na+/Cl- cotransporters, hav e been prepared to facilitate the identification and characterization of SERT proteins in native and transfected cells. Immunoprecipitations and immunoblots of rat/human SERT-transfected HeLa cells reveal speci fic SERT-immunoreactive glycoproteins absent from extracts of vector-t ransfected cells and absent when incubations were conducted using pept ide- or fusion protein-absorbed antibody. In SDS-PAGE of membranes pre pared from rat midbrain and cortex, SERTs migrate as single 76 kDa pol ypeptides with a relative abundance consistent with the known distribu tion of 5HT neurons and axonal projections. SERT-immunoreactive protei ns are also detectable in platelet and pulmonary membranes, sites of p eripheral 5HT uptake, but not in liver. Our studies also indicate that brain and platelet SERTs are formed from identical polypeptides diffe ring significantly in their extent of N-linked glycosylation. Immunocy tochemistry performed on rat brain sections with CT-2 antibody reveale d SERT expression associated with brainstem raphe nuclei in a pattern virtually identical to that obtained by labeling adjacent sections wit h 5HT antisera. SERT-immunoreactive fibers were found to be widely dis tributed throughout the rodent brain, with highest density in forebrai n regions known to receive a dense serotonergic innervation. in a simi lar manner, CT-2 antibody also detects endogenous expression of human SERT proteins, providing an opportunity for future studies on the modu lation of transporter protein expression in neurologic and psychiatric disorders.