Antibodies against the extracellular domain of the 5-HT 3 receptor label both native and recombinant receptors

Abstract

We have developed polyclonal antibodies (pAb120) against a peptide corresponding to a region within the extracellular domain of the 5-hydroxytryptamine 3 (5-HT 3) receptor subunit, thus permitting, for the first time, localization of 5-HT 3 receptors at the cell surface in intact (non-permeabilized) systems. The antibodies are both specific and sensitive: pAb120 recognized as little as 63 ng of protein from HEK293 cells expressing recombinant 5-HT 3 receptors, whilst Western blots of recombinant 5-HT 3 receptors purified from Sf9 cells revealed two bands at 48 and 54 kDa, and native 5-HT 3 receptors from N1E-115 cell membranes produced a broad band at 50–54 kDa with a smaller band at 35 kDa. These bands were also labelled by antibodies against the intracellular loop of the 5-HT 3 receptor. Immunofluorescent labelling revealed a ring of intense fluorescence in the plasma membrane of non-permeabilized HEK293 cells expressing recombinant 5-HT 3 receptors. Studies on native 5-HT 3 receptors revealed that pAb120 could recognize 5-HT 3 receptors on presynaptic terminals isolated from rat striatum, and immunohistochemical studies in rat brain sections revealed labelling of cell bodies, dendrites and varicose axons in hippocampus, cortex and lateral hypothalamus; all of these areas have been reported to express 5-HT 3 receptors. We conclude that pAb120 is a highly specific and sensitive antiserum that will assist in clarifying fundamental questions about 5-HT 3 receptor neurobiology.