The Arginine Taste Receptor: Physiology, Biochemistry, and Immunohistochemistry^a

Abstract

The amino acid, L-arginine (L-Arg), is a potent taste stimulus for the channel catfish, Ictalurus punctatus. Receptor binding studies demonstrated a high-affinity binding of L-Arg to putative taste receptor sites. This binding could be inhibited by preincubation of the tissue in the lectins Phaseolus vulgaris agglutinin (PHA) and Ricinus communis agglutinin I (RCA I). Neurophysiological studies demonstrated that the L-Arg receptor is a stimulus-gated ion channel type receptor whose conductance was stimulated by L-Arg and inhibited by D-arginine (D-Arg). To purify the receptor we subjected CHAPS solubilized partial membrane preparation from barbel epithelium to RCA I lectin affinity chromatography. The bound proteins were eluted with D-galactose. When these proteins were reconstituted into lipid bilayers, L-Arg activated single channel currents with conductances between 45 and 85 pS. Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the eluted protein showed a distinct band at approximately 83 kDa. Polyclonal antibodies raised against this 83-kDa band in guinea pigs reacted with numerous small (approximately 1 micron) sites within the taste pore of every taste bud when applied to fixed nonpermeabilized barbels. This observation suggests that the antibodies recognize an externally-facing epitope of the putative Arg receptor. The antibodies also inhibited L-Arg-stimulated currents in reconstitution studies. Sephacryl S-300 HR chromatography of the eluant from the affinity column showed a high molecular weight peak (> 700 kDa) which was recognized by the antibodies. Reconstitution of the protein from this peak into a lipid bilayer resulted in L-Arg-stimulated channels that could be inhibited by D-Arg. This high molecular weight component may be aggregates of the arginine taste receptor.

Untitled

“…Preliminary reports on the localization of an L-ArgR showed that the antibodies, GP1, and the lectin, PHA-E, when incubated with intact, unfixed barbels, labelled exterior-facing epitopes on catfish barbel taste buds [35,45] and SCCs scattered in the epithelium among taste buds [35]. Immunoelectron microscopy using GP1 revealed labelling primarily on those cells of the taste bud containing large microvilli [35].…”

“…The procedure for development of antibodies against the catfish barbel peptides labelled by the lectins, RCA-I and PHAE has been described previously [45]. Briefly, electroeluted material from that area of a gel congruent with an identical gel labelled by the lectins was injected into three female guinea pigs using a schedule and procedure previously found to raise high titer polyclonal antibodies [60].…”

“…In addition, PHA-E also labelled the apical membrane of selected taste bud cells and solitary chemoreceptor cells of catfish barbel [35]. When reacted against the exterior epithelium of fixed, unpermeabilized catfish barbel, polyclonal antibodies developed against these lectin reactive peptides immuno-labelled the apical membrane of a subset of barbel taste receptor cells [45]. …”

Chemical Senses

“…l-Arginine stimulates specific reconstituted channels, which have been localized to the pores of guinea-pig taste buds (Grosvenor et al, 1998).…”