D. Lynn Kalinoski

Differential interaction of lectins with chemosensory receptors

L-Alanine and L-arginine bind with similar affinity (Kd 10(-7)-10(-6) M) to receptors in both a sedimentable fraction (P2) from taste epithelium and isolated olfactory cilia from the channel catfish, Ictalurus punctatus. Lectins of differing carbohydrate specificity were used to determine the glycoprotein nature of the chemosensory plasma membranes and to differentially affect receptors for L-alanine and L-arginine. The peroxidase-conjugated lectins concanavalin A (Con A), wheat germ agglutinin (WGA), and peanut agglutinin (PNA) were used to identify the glycoprotein components of the chemosensory plasma membranes after polyacrylamide gel electrophoresis. In both chemosensory membranes, numerous protein components were labelled by Con A and WGA. In contrast, a single predominant component was labeled by PNA in olfactory cilia, whereas several proteins in taste membranes were labeled by this lectin. When unconjugated lectins were preincubated with olfactory cilia, 60-70% of binding to L-alanine and L-arginine receptors was inhibited by Con A and WGA. PNA inhibited L-alanine but not L-arginine binding to olfactory receptors. Inhibition of olfactory receptor binding by lectins was time- and dose-dependent. By contrast, no inhibition of either L-alanine or L-arginine receptor binding in taste membranes was observed with any of the lectins. The differential labeling of the chemosensory membranes and the differential inhibition of receptor binding by lectins suggest that, despite ligand similarity, the chemosensory receptors in these membranes are not identical molecular species.

Specificl-arginine taste receptor sites in the catfish, Ictalurus punctatus: biochemical and neurophysiological characterization

We report here the characterization of the arginine binding site(s) and corroborative neurophysiological studies. Binding of L-[3H]arginine to Fraction P2 from taste epithelium was measured by a modification of the method of Krueger and Cagan. Parameters for measuring maximal binding activity were established for both duration of incubation and pH of medium. At pH 7.8, the apparent single rate constant for association (kobs) at 4 degrees C was 4.72 x 10(+5).M-1.min-1. Dissociation was more complex, yielding two rate constants of 1.77.min-1 and 8.34 x 10(-3).min-1. These data suggest the presence of two affinity states for L-arginine. The KD values as calculated from the ratio k-1/k+1 were 1.3 x 10(-6) M and 1.8 x 10(-8) M. Homologous inhibition studies of L-arginine binding were not fit by a simple mass action relationship (Hill Coefficient 0.79), but were best fit by a two-site model with IC50 values of 1.6 x 10(-6) M for the high affinity state and 9 x 10(-4) M for the low affinity state. Multiunit neural recordings examined the stimulatory effectiveness of a number of guanidinium-containing compounds. Compared with L-arginine, only L-arginine methyl ester and L-alpha-amino-beta-guanidino propionic acid (L-AGPA) were effective stimuli. Cross-adaptation experiments demonstrated that at 10(-4) M L-arginine methyl ester, L-AGPA and, to a lesser extent, D-arginine were effective cross-adapting stimuli to 10(-6) M L-arginine. In competition binding studies L-arginine methyl ester, L-AGPA and D-arginine also inhibited binding of L-[3H]arginine (10(-6) M), but each recognized only one affinity state. Inhibition by the poorly cross-adapting stimuli L-glutamate, glycine and L-alanine occurred only above 10(-3) M, indicating that the binding sites for L-arginine are selective. These studies suggest that there are at least two affinity states of L-arginine binding, that the binding sites are specific, and that effective agonists of L-arginine receptors must contain a guanidinium group and an unblocked L-alpha-amino group.

Biochemical studies of taste sensation. XIII. Enantiomeric specificity of alamine taste receptor sites in catfish,Ictalurus punctatus

Specific binding of amino acid taste stimuli is known to occur to a sedimentable fraction (P2) from catfish (Ictalurus punctatus) taste epithelium or to purified plasma membranes from that fraction. L-Alanine, a potent taste stimulus for the catfish, binds in a reversible and saturable manner to these preparations. The extent to which the enantiomeric stimuli, L- and D-alanine, interact with the same or different receptor/transduction processes is investigated here both electrophysiologically and biochemically. With an electrophysiological assay, L-alanine was the more potent stimulus across a concentration range of 10(-9)-10(-3) M, yet both enantiomers displayed approximately the same threshold. The concentration-electrophysiological response functions for each enantiomer were different. That of L-alanine was approximately linear across the (log) concentration range while that of D-alanine was non-linear, with small but definitely observable responses being noted from 10(-9)-10(-5) M D-alanine, and larger incremental responses thereafter. With most of the nerve bundle preparations studies, L- and D-alanine cross-adapted one another, but this cross-adaptation was not always complete. Experiments in which both L- and D-alanine were present in a 1:1 mixture of equally stimulatory concentrations suggested the existence of receptor or transduction processes unique to each enantiomer. Biochemically binding studies demonstrated high affinity binding sites for both enantiomers with values of Kd-app for L-alanine of 1.5 microM and for D-alanine of 25 microM. For both enantiomers, additional lower-affinity binding sites were observable. The capacity of the lower-affinity sites was particularly great for D-alanine. The enantiomers competed one with the other for binding, with L-alanine showing greater competitive ability than D-alanine at low concentrations. For the high affinity sites, double-reciprocal plots of the data suggested a competitive mechanism. The lower affinity sites for D-alanine were less accessible to L-alanine compared with the high affinity sites of D-alanine. Both the biochemical and electrophysiological results indicate that while a portion of the responses to L- and D-alanine occurs through a common receptor/transduction process, there exist independent receptor/transduction processes for the enantiomers, L- and D-alanine.

Use of Monoclonal Antibodies to Characterize Amino Acid Taste Receptors in Catfish

One approach to investigating the specificity of taste receptor sites is through the use of specific, site-directed agents. Because antibodies are ideally suited for this, we previously developed monoclonal antibodies that interact with catfish taste epithelial plasma membranes and inhibit the in vitro binding of L-alanine.’ The interaction of several of these antibodies with two putative amino acid taste receptors, the alanine and arginine receptors, has been further characterized with respect to the specificity of binding inhibition, their effect on neural responses, and the identity of the antigen(s). Monoclonal antibodies from two clones, termed G-7 and G-10, have been used in these studies following purification on Protein A-Sepharose. Both antibodies inhibited the binding of r-[’HH]alanine by the plasma membrane fraction (Fraction P2) of catfish taste epithelium at antibody protein concentrations ranging from 0.5 to 12 pg/ml. Controls such as bovine serum albumin (BSA) and nonimmune mouse IgG did not inhibit alanine binding. G10 antibody also inhibited the binding of ~-[’H]arginine from 19.455.5%, depending on the concentration of ligand and antibody; inhibition decreased with higher concentration of arginine and lower concentration of antibody. This inhibition is similar in magnitude to the inhibition of L-alanine binding described earlier.’ In a neurophysiological assay, antibodies G-7 and G-10 exhibited slight but sustained excitatory activity when applied to the catfish taste epithelium, while the controls, BSA and nonimmune mouse IgG, did not. Variable inhibition (520%) of

Strategies for Isolation of Taste Receptor Proteins

For almost two decades the channel catfish, Ictalurus punctatus, has proved a useful model for the study of the vertebrate gustatory system. During this period a combination of behavioral [1], neurophysiological [2–4], and biochemical [5–7] techniques have been directed at identifying specific taste receptors and characterizing the molecular mechanisms underlying sensory transduction mediated by these receptors. Neurophysiological experiments have established the presence of independent receptor sites for l-alanine (and possibly other short-chain neutral amino acids) [2,3], l-arginine [2,3], and l-proline [2–4]. In addition, receptors sites for some d-isomers of amino acids, in particular d-alanine and d-arginine, have been inferred from non-reciprocal cross-adaptation obtained in electrophysiological studies [3]. Receptor-binding methodologies utilizing both equilibrium and kinetic techniques have been employed to determine the apparent dissociation concentrations for the l-alanine (1.5μM) [5,6] and l-arginine (18 nM and 1.3μM) [7] binding sites.

Enantiomeric Specificity of Alanine Taste Receptor Sites in Catfish

The mechanisms underlying discrimination of enantiomers by taste receptors are little understood. The cutaneous taste system of the catfish (Ictalurus punctatus) lends itself to analysis of enantiomeric discrimination because this system is differentially sensitive to enantiomers of amino acids.’ Using this animal model, the molecular details of enantiomeric discrimination in taste can be studied both biochemically and electrophysiologically. Specific binding of stimulus amino acids has been demonstrated to receptor-containing membrane preparations from catfish taste epithelium?“ Discrimination of the enantiomeric stimuli, Land D-alanine, by the catfish cutaneous taste system has been investigated here using combined biochemical and electrophysiological approaches. The electrophysiological assay showed that both Land D-alanine stimulated the facial nerve when aqueous solutions of the stimuli flowed over the barbel, but that the relative stimulatory abilities of the enantiomers differed. Responses to L-alanine increased more sharply with concentration to lo-’ M) than did those to Dalanine to lo-’ M ) , even though the threshold values for both enantiomers appeared to be nearly equal to lo-* M ) . With most of the nerve bundle preparations studied, Land D-alanine at equally stimulatory concentrations crossadapted one another, but this cross-adaptation was not always complete. Additional experiments in which Land D-alanine were present in a 1 : 1 mixture of equally stimulatory concentrations usually evoked a larger magnitude of response than did each single component alone. These results from cross-adaptation and mixture studies are consistent with the hypothesis that at least two populations of alanine-responsive transduction pathways are present. This hypothesis was examined at the receptor level using a biochemical binding assay that measured the specific binding of the enantiomers to receptor tissue and that detected binding competition between the enantiomers. The enantiomers, Land

Role of Inositol Triphosphate (IP 3 ) in Olfactory Transduction

Olfactory neurons respond to stimulation with odorants by increasing the frequency of action potential discharge. The sequence of biochemical reactions that culminates in action potential discharge is believed to start with the interaction of odor stimuli with specific G-protein-coupled receptors located on the plasma membrane of cilia that extend into the mucous layer. Families of putative odorant receptors have been identified in several species [1–3], and a putative receptor clone has been shown to cause stimulation of second messenger formation when expressed in a heterologous system [4].