Biochemical enrichment and biophysical characterization of a taste receptor for L-arginine from the catfish, <it>Ictalurus puntatus</it>

• Main Authors: Spielman Andrew I, Kaulin Yuri, Grosvenor William, Bayley Douglas L, Kalinoski D Lynn, Teeter John H, Brand Joseph G
• Format: Article
• Language: English
• Published: BMC 2004-07-01
• Series: BMC Neuroscience
• Subjects: Chemical senses; Taste; Signal transduction; Lectin; Ion channel; Receptor; Immunohistochemistry; Protein purification; Lipid bilayer
• Online Access: http://www.biomedcentral.com/1471-2202/5/25

<p>Abstract</p> <p>Background</p> <p>The channel catfish, <it>Ictalurus punctatus</it>, is invested with a high density of cutaneous taste receptors, particularly on the barbel appendages. Many of these receptors are sensitive to selected amino acids, one of these being a receptor for L-arginine (L-Arg). Previous neurophysiological and biophysical studies suggested that this taste receptor is coupled directly to a cation channel and behaves as a ligand-gated ion channel receptor (LGICR). Earlier studies demonstrated that two lectins, <it>Ricinus communis </it>agglutinin I (RCA-I) and <it>Phaseolus vulgaris </it>Erythroagglutinin (PHA-E), inhibited the binding of L-Arg to its presumed receptor sites, and that PHA-E inhibited the L-Arg-stimulated ion conductance of barbel membranes reconstituted into lipid bilayers.</p> <p>Results</p> <p>Both PHA-E and RCA-I almost exclusively labeled an 82–84 kDa protein band of an SDS-PAGE of solubilized barbel taste epithelial membranes. Further, both rhodamine-conjugated RCA-I and polyclonal antibodies raised to the 82–84 kDa electroeluted peptides labeled the apical region of catfish taste buds. Because of the specificity shown by RCA-I, lectin affinity was chosen as the first of a three-step procedure designed to enrich the presumed LGICR for L-Arg. Purified and CHAPS-solubilized taste epithelial membrane proteins were subjected successively to (1), lectin (RCA-I) affinity; (2), gel filtration (Sephacryl S-300HR); and (3), ion exchange chromatography. All fractions from each chromatography step were evaluated for L-Arg-induced ion channel activity by reconstituting each fraction into a lipid bilayer. Active fractions demonstrated L-Arg-induced channel activity that was inhibited by D-arginine (D-Arg) with kinetics nearly identical to those reported earlier for L-Arg-stimulated ion channels of native barbel membranes reconstituted into lipid bilayers. After the final enrichment step, SDS-PAGE of the active ion channel protein fraction revealed a single band at 82–84 kDa which may be interpreted as a component of a multimeric receptor/channel complex.</p> <p>Conclusions</p> <p>The data are consistent with the supposition that the L-Arg receptor is a LGICR. This taste receptor remains active during biochemical enrichment procedures. This is the first report of enrichment of an active LGICR from the taste system of vertebrata.</p>