Biochemical enrichment and biophysical characterization of a taste receptor for L-arginine from the catfish, Ictalurus puntatus.
BACKGROUND
The channel catfish, Ictalurus punctatus, 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, Ricinus communis agglutinin I (RCA-I) and Phaseolus vulgaris 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.


RESULTS
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.


CONCLUSIONS
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.
Grosvenor
William
W
Monell Chemical Senses Center, Philadelphia, PA 19104-3308, USA. william.grosvenor@worldnet.att.net
Kaulin
Yuri
Y
Spielman
Andrew I
AI
Bayley
Douglas L
DL
Kalinoski
D Lynn
DL
Teeter
John H
JH
Brand
Joseph G
JG
D000180:Acrylic Resins / Q000737:chemistry; D000818:Animals; D000838:Anions / Q000378:metabolism; D001120:Arginine / Q000378:metabolism* / Q000502:physiology; D001703:Biophysics / Q000379:methods*; D002412:Cations / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D003530:Cyprinidae / Q000276:immunology; D007059:Ictaluridae / Q000235:genetics*; D007106:Immune Sera / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods; D015640:Ion Channel Gating / Q000502:physiology; D037102:Lectins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D008051:Lipid Bilayers / Q000378:metabolism; D008564:Membrane Potentials / Q000502:physiology; D037121:Plant Lectins / Q000737:chemistry; D020673:Protein Renaturation; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000378:metabolism*; D013650:Taste Buds / Q000737:chemistry*
D000180:Acrylic Resins; D000838:Anions; D002412:Cations; D007106:Immune Sera; D037102:Lectins; D008051:Lipid Bilayers; D037121:Plant Lectins; D043562:Receptors, G-Protein-Coupled; C017707:Ricinus communis agglutinin-1; C037239:Sephacryl Superfine; C431926:taste receptors, type 1; D001120:Arginine