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.
Monell Chemical Senses Center, Philadelphia, PA 19104-3308, USA. william.grosvenor@worldnet.att.net
William
W
Grosvenor
Yuri
Y
Kaulin
Andrew I
AI
Spielman
Douglas L
DL
Bayley
D Lynn
DL
Kalinoski
John H
JH
Teeter
Joseph G
JG
Brand
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