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

BMC Neurosci. 2004 Jul 28;5:25. doi: 10.1186/1471-2202-5-25.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acrylic Resins / chemistry
  • Animals
  • Anions / metabolism
  • Arginine / metabolism*
  • Arginine / physiology
  • Biophysics / methods*
  • Cations / metabolism
  • Chromatography, Affinity / methods
  • Chromatography, Gel / methods
  • Chromatography, Ion Exchange / methods
  • Cyprinidae / immunology
  • Ictaluridae / genetics*
  • Immune Sera / metabolism
  • Immunohistochemistry / methods
  • Ion Channel Gating / physiology
  • Lectins / chemistry
  • Lectins / immunology
  • Lectins / metabolism
  • Lipid Bilayers / metabolism
  • Membrane Potentials / physiology
  • Plant Lectins / chemistry
  • Protein Renaturation
  • Receptors, G-Protein-Coupled / chemistry*
  • Receptors, G-Protein-Coupled / metabolism*
  • Taste Buds / chemistry*

Substances

  • Acrylic Resins
  • Anions
  • Cations
  • Immune Sera
  • Lectins
  • Lipid Bilayers
  • Plant Lectins
  • Receptors, G-Protein-Coupled
  • Ricinus communis agglutinin-1
  • Sephacryl Superfine
  • taste receptors, type 1
  • Arginine