Use of the channel catfish for these studies was approved by the Institutional Animal Care and Use Committee of the Monell Chemical Senses Center. Channel catfish, Ictalurus punctatus, purchased from local suppliers were usually euthanized on the day of delivery, but if not, were held less than 4 days in 250 gallon aquaria under dim light and fed commercial catfish chow.
All electrolytes, buffers and other chemicals were reagent grade from Sigma (St. Louis, MO). Water was deionized and further purified through a Milli-Q Plus PF system (Bedford, MA). 3-[(3-Cholamidopropyl)-dimethylammonio]-1-propanesulfonate (98%) (CHAPS), polyoxyethylenesorbitan monolaurate (TWEEN 20), phenylmethanesulfonyl fluoride (PMSF) and pepstatin A were purchased from Sigma. n-Octyl-β-D-gluco-pyranoside (n-octylglucoside) was purchased from Calbiochem (LaJolla, CA). The absolute enantiomer, L-arginine HCl, was purchased from Sigma, and the absolute enantiomer, D-arginine HCl, was a gift of the Ajinomoto Co., Tokyo, Japan. Sephacryl S-300 HR, High Range Gel Filtration Calibration Kits, and 1.0 ml Hitrap Q columns were purchased from Pharmacia Biotech (Piscataway, NJ). Agarose-bound lectins, Ricinus communis agglutinin I (RCA-I), Phaseolus vulgaris Erythroagglutinin (PHA-E), in their biotinylated forms, the ABC kits, and rhodamine-conjugated RCA-I were purchased from Vector Lab (Burlingame, CA). The second antibody, Cy3-conjugated goat anti-guinea pig IgG, was obtained from Jackson ImmunoResearch Labs. (West Grove, PA). The 4 CN Membrane Peroxidase Substrate System was purchased from Kirkegaard & Perry Laboratories (Gaithersburg, MD). Gels and ampholytes were purchased from BioRad (Hercules, CA). Protein was quantitated using a BioRad DC Protein Assay. Synthetic 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) were purchased from Avanti Polar Lipids, Inc. (Pelham, AL).
Tissue homogenate preparation and solubilization
Maxillary and mandibular barbels from approximately 50 euthanized channel catfish, ~25–40 cm long, were removed and placed in a beaker containing 100 ml of 50 mM Tris-HCl buffer (pH = 7.8), 100 mM NaCl, 1 mM EDTA ("TRIS Wash"). After one exchange of buffer, barbels were removed to a 50 ml polypropylene screw cap tube filled with TRIS Wash buffer and stored at -80°C until used.
The preparation of a solubilized, plasma membrane-enriched fraction from catfish barbels was adapted from Kalinoski . A typical homogenate/solubilization run used barbel tissue from 150 fish, prepared in aliquots of 50 fish at a time. The entire procedure was carried out at 4°C. The epithelium of thawed barbels from 50 fish at a time in 75 ml of TRIS Wash was stripped from the supporting pseudo-cartilage with two 10 second bursts (with a 10 second interval) from a hand-held Toastmaster Hand Blender, Model 1738 (Boonville, MO). The suspension was allowed to settle, and the supernatant decanted into a 600 ml beaker through two layers of USP Type VII gauze (Kendall Co., Boston, MA). Fifty ml of TRIS Wash (4°C) was added to the remaining settled barbels, and the suspension subjected to a third 10 second burst from the Blender. This entire second suspension was rapidly poured over the gauze layers into the same beaker. A second and third tube of barbels from 50 fish were treated identically, and the filtrate from all three combined, and the volume brought to 470 ml. This homogenate, divided into two aliquots, was centrifuged at 4000 × g for 15 min. The supernatants were recovered and centrifuged at 21,500 × g for 45 min. The pellets were retained.
To solubilize the pellets from the 21,500 × g spin each pellet was recovered by two, 1 ml rinses of 50 mM TrisHCl (pH 7.8), 50 mM NaCl ("Low Osmolar Buffer") (total, 4 ml) and transferred to a 15 ml Teflon/glass homogenizer. Twenty milligrams of CHAPS and 40 μl of protease inhibitor-mix (0.275 mM pepstatin A, 57.5 mM PMSF, in ethanol) were added to the ~4 ml suspension in the homogenizer. The suspension was homogenized by ten slow strokes of the Teflon pestle using a rotating motor drive at moderate speed. The homogenate was transferred to a 15 ml capped tube, diluted to 10 ml with Low Osmolar Buffer, and placed on a Clay Adams Nutator rocker/shaker overnight at 4°C to solubilize the proteins. After the overnight agitation, the suspension was centrifuged for 1 h at 100,000 × g. The supernatant was recovered and is referred to as "Sp." This Sp was then used in the subsequent lectin affinity chromatography step.
Lectin affinity chromatography
All steps in the lectin affinity procedure were performed at 4°C. Agarose-bound RCA I affinity resin was pre-washed by adding 8 ml of gel slurry to a 125 ml conical glass tube and bringing the final volume to 14 ml with TRIS Wash. The tube was inverted several times, then centrifuged at 500 × g for 1 min. The supernatant was discarded and the wash step repeated three additional times with TRIS Wash buffer, then two more times with Low Osmolar buffer. The resulting agarose-bound RCA I gel, with a bed volume of 4 ml, was used for affinity chromatography.
The 4 ml agarose-RCA gel in the 15 ml tube was combined with ~7 ml of Sp, and the mixture equilibrated on the Nutator tube rocker for 30 min. The mixture was poured into a 1 cm × 10 cm column and the effluent collected as 1 ml fractions at a flow rate of 6.0 ml/h. Effluent from the column was monitored by absorbance at 230 nm and/or 280 nm.
To remove unbound protein, the column was washed with a sufficient volume (~12 ml) of Low Osmolar Buffer until no detectable protein eluted from the column. Proteins bound to the RCA resin were then eluted from the column with 10 ml of 50 mM Tris-HCl (pH = 7.8), 200 mM NaCl, 1 mM EDTA, 20 mM D-galactose. Protein from both the Low Osmolar elution step and the D-galactose elution step was reconstituted into lipid bilayers and assayed for L-Arg-stimulated ion channel activity (See ahead).
The galactose-eluted protein fractions showing L-Arg-stimulated ion channel activity were pooled and dialyzed over night against 2000 ml Milli-Q water containing 0.05% CHAPS. Dialyzed samples were lyophilized and stored at -80°C.
Gel filtration chromatography
All steps in the gel filtration procedure were performed at 4°C. Lyophilized proteins from the galactose elution of the lectin column were prepared for further enrichment using size exclusion chromatography by dissolution in 300 μl of TRIS Wash with the addition of 0.2% CHAPS and 10% sucrose. This preparation was loaded onto a Sephacryl S-300 HR column (1 cm × 40 cm). Fractions of 0.4 ml were eluted from the column with a buffer composed of TRIS Wash plus 0.2% CHAPS. The column was run at 2.4 ml/hr, and the effluent absorbance monitored at 230 nm and 280 nm. Eluted fractions containing measurable protein were evaluated for L-Arg-stimulated ion channel activity by incorporation into a lipid bilayer (See ahead). In addition, SDS-PAGE (See ahead) was performed on each eluted fraction. Those fractions exhibiting L-Arg-stimulated ion channel activity were dialyzed overnight against 2000 ml of Milli-Q water containing 0.05% CHAPS, lyophilized and stored at -80°C.
The column was calibrated with a Gel Filtration Calibration Kit with protein standards from 158 to 669 kDa.
Ion exchange chromatography
All steps in the ion exchange procedure were performed at 4°C. Further enrichment of the fractions exhibiting L-Arg-stimulated ion activity was achieved using an anion exchange column (Hitrap Q). The column was prepared by washing with 5.0 ml of Start Buffer (25 mM Tris-HCl, pH = 9.0), followed by 2.0 ml of Regeneration Buffer (Start Buffer plus 1.0 M NaCl) and then 5.0 ml of Start Buffer. Lyophilized proteins of the active fraction from the gel filtration column were dissolved in 300 μl of Start Buffer and applied to the column. Prior to elution, the loaded column was washed with 2.0 ml Start Buffer. Elution was at 12 ml/h in 400 μl fractions. First, 1.0 ml of First Elution Buffer (25 mM Tris-HCl, pH = 9.0, 500 mM NaCl) was applied to the column followed by (second), 2.0 ml of Start Buffer (no NaCl), followed by (third) 4.0 ml of Second Elution Buffer (25 mM Tris-HCl, pH = 8.0, 500 mM NaCl). Effluent from the column was monitored with in line UV (230 and 280 nm) and conductivity detectors. Each fraction was assayed for L-Arg-stimulated activity (See ahead.). The active fractions were pooled, dialyzed overnight against 2000 ml of Milli-Q water containing 0.05% CHAPS, lyophilized, and stored at -80°C.
Development of polyclonal antibodies
The procedure for development of antibodies against the catfish barbel peptides labelled by the lectins, RCA-I and PHAE has been described previously . Briefly, electroeluted material from that area of a gel congruent with an identical gel labelled by the lectins was injected into three female guinea pigs using a schedule and procedure previously found to raise high titer polyclonal antibodies . Antisera were aliquoted into 500 μl lots in 1.5 ml Eppendorf tubes and kept frozen at -80°C until used. Antisera from animal #1 (GP1) and animal &2 (GP2) were found to be the most specific in that they reacted primarily with their antigen within the 82–84 kDa band in Western blots of SDS-PAGE of catfish barbel membranes.
The IgG fraction of GP1 and GP2 antisera was purified using an E-Z-SEP antibody purification kit (Pharmacia). To reduce non-specific binding in the immunohistochemical studies and Western blots, the GP1 and GP2 antibodies were incubated with powder derived from an acetone precipitation of a catfish brain homogenate. One ml of E-Z-SEP-purified antibody was incubated with 10 mg of powder for 40 min at 4°C. The powder was removed by centrifugation and the procedure was repeated once with fresh powder. The resulting pretreated antibodies are called simply, "GP1" and "GP2."
Gel electrophoresis, lectin blots and Western blots
Tris-glycine gels (4–20%, Bio-Rad) were used for SDS-PAGE. Protein of fractions before any chromatography (i.e., Sp), as well as those from each chromatography step, were denatured by mixing 10 μl from each fraction, 1:1, with sample buffer containing 125 mM Tris-HCl (pH = 8.0), 20% glycerol, 4% SDS, 4% β-mercaptoethanol and 50 μg/ml bromophenol blue, and placing the mix in a boiling water bath for 5 min. Gels were run at a constant 20–25 mA for about 1 h, using prestained broad range molecular weight markers (Bio-Rad) in 1 or more lanes. Proteins were stained with Bio-Rad Silver Stain Plus kit.
For lectin blots, proteins were electrophoretically transferred to nitrocellulose sheets (Bio-Rad Mini Trans-Blot, Hercules, CA). The sheets were incubated in a blocking solution (2% gelatine, phosphate buffered saline [(PBS) (150 mM NaCl, 100 mM sodium phosphate, pH = 7.4)] and 0.05% Tween 20) for 2 h at room temperature, and then incubated with biotinylated RCA-I or PHA-E (10 μg/ml) overnight at 4°C. Following exposure to biotinylated lectin, nitrocellulose sheets were washed extensively with blocking solution. Lectin-bound protein bands were visualized using a Vectastain peroxidase ABC kit with a 4 CN Membrane Peroxidase Substrate system. Development was stopped after one hour using 5% glacial acetic acid.
For Western blots, proteins were transferred to nitrocellulose and incubated in blocking solution (PBS, pH = 7.4, 5% non-fat dry milk, 1% goat serum and 0.05% TWEEN 20) for 2 h at ambient temperature with constant slow rocking (Nutator). The nitrocellulose was incubated overnight at 4°C (rocking) with primary antibody (GP1, 1/500) in blocking solution. The nitrocellulose was washed with blocking solution and incubated with biotinylated secondary antibody (1:250) for 1 h with slow rocking. Bands were visualized as above.
Lectin histochemistry and immunohistochemistry
Rhodamine-conjugated RCA-I (Vector Labs) was used to estimate the specificity of lectin interaction with glycoproteins of catfish barbel. To assess the localization of the antigen contained in the 82–84 kDa band from the SDS-PAGE of a membrane fraction of catfish barbel (and thereby the likely localization of the putative taste receptor for L-Arg), immunohistochemistry was performed using the GP1 and GP2 antibodies pretreated as described above.
Barbels were removed from euthanized albino channel catfish (I. punctatus) (the fish being 5 – 7 cm in length) and immediately placed in 4% buffered paraformaldehyde (PFA) (0.1 M sodium phosphate buffer, pH 7.2–7.4) for eight hours at 4°C. After washing out the PFA with several rinses of excess buffer, the barbels were placed successively in 10%, 20%, and 30% sucrose (in buffer) for 24 hr, all at 4°C. After the final cryoprotect sucrose step, barbels were cut into pieces of less than 1 cm and mounted with M-1 Embedding Matrix (Thermo Shandon, Pittsburgh, PA). The tissue was sectioned at 10 microns on a Microm HM500OM cryostat.
For lectin histochemistry, ten micron sections of fixed barbel were incubated in the dark with rhodamine-conjugated RCA-I lectin (Vector Labs., Burlingame, CA), diluted 1/200 for 2 to 3 hr at ambient temperature. The sections were then washed quickly with Dulbecco's PBS (GIBCO/Invitrogen Corp), followed by three incubation washes of 10 min each.
For immunohistochemistry, 10 micron barbel sections, pre-washed 3 times for 10 min each in Dulbecco's PBS, were first incubated at ambient temperature for 3 to 5 hr in blocking buffer consisting of 3% bovine serum albumin, 2% goat serum, 0.3% TritonX100, and 0.1% sodium azide in Dulbecco's PBS at pH 7.1. The sections were then incubated with primary antibody, GP1 or GP2, in blocking buffer for 18 hr at 4°C. The primary antibody solution was removed and the sections were then washed once quickly with Dulbecco's PBS, followed by three incubation washes with PBS of 10 min each. The sections were then incubated in the dark with second antibody, Cy3-conjugated goat anti-guinea pig IgG (Jackson ImmunoResearch Labs., West Grove, PA) at 1:1000 dilution for 60 min. at ambient temperature. The secondary antibody was removed and the sections washed once quickly with Dulbecco's PBS, followed by three incubation washes with PBS of 10 min each. Excess fluid was removed from the slides and the sections mounted under cover slips with VectaShield (Vector Labs). Sections were observed with a Nikon Microphot FXA fluorescence microscope, photographed, and images sized and enhanced using the GNU Image Manipulation Program software .
Immuno-specificity was verified by running negative controls where the primary antibody was omitted from the procedure. In all cases, this control step showed no taste bud labelling (Fig. 5E). The scattered, spotty background labelling seen with the primary antibodies is likely due to both an unknown factor in pre-immune serum and to labelling of solitary chemoreceptor cells in the barbel epithelium, as was previously documented .
Lipid bilayer reconstitution
Reconstitution of protein fractions containing likely L-Arg-stimulated channel activity was carried out with material from the low osmolar and galactose-eluted fractions of the lectin affinity procedure, from the protein-containing fractions off the Sephacryl S-300 gel filtration column and from each fraction of the ion exchange column. Lipid vesicles were prepared by sonication of 5 mg of DOPE:DOPC (2:1) and 0.5 ml of 5 mM Tris-HCl (pH = 7.2), 300 mM NaCl and 500 mM sucrose, to which 10 μg n-octylglucoside was added. To prepare the liposome-detergent mixture for incorporation into a lipid bilayer, approximately 0.2 to 0.5 μg of presumed receptor protein was added to the liposome in a cassette dialysis unit, and the mixture dialyzed overnight against 2000 ml of the Tris/NaCl/sucrose buffer at 4°C.
Virtually solvent-free lipid bilayer membranes were prepared as described . The membrane-forming solution was an equimolar mixture of DOPS:DOPE in hexane. The bilayer chamber consisted of two symmetrical halves of a Teflon chamber, each with solution volumes of 1 ml divided by a 15 μm thick Teflon partition containing a round aperture of about 150 μm diameter. Hexadecane in n-hexane (1:10, v/v) was used for aperture pre-treatment. A pair of Ag-AgCl electrodes was connected to the solution in the chamber via 3 M KCl-4% agar bridges. "Virtual ground" was maintained at the trans side of the bilayer.
The bilayer was bathed symmetrically with 5 mM MOPS (pH 7.2), 1 mM CaCl2, 100 mM NaCl (unless otherwise stated). Fifty to 100 μl of the dialyzed liposome vesicles containing presumed receptor was added to the cis-side of the membrane. Fusion of the vesicles was initiated mechanically by gently mixing the membrane bathing solution from the cis-side using a micro-pipette. L-Arg was added to the cis side approximately 20 min after addition of vesicles. Unless otherwise stated all other additions of reagents also were made from the cis side. Channel sidedness was determined by sensitivity of the bilayer to L-Arg. The orientation of the channels was such that the L-Arg sensitive side was normally in the cis compartment. All bilayer experiments were performed at room temperature.
The current was amplified by a Dagan 3900 integrating patch-clamp amplifier (Dagan Corp., Minneapolis, MN) in the voltage clamp mode. Single channel data were digitized at 15 kHz (Digidata 1200, Axon Instruments, Foster City, CA) and analyzed using pClamp6 (Axon Instruments) and Origin 5.1 (Microcal Software, North Hampton, MA) software on an IBM compatible computer.
The calculated success rate of incorporation of vesicular proteoliposomes into lipid bilayers was about 25%. Success rate is defined as the ratio of the number of successful incorporation attempts to the total number of incorporation attempts (an "incorporation attempt" refers here to the formation of a new bilayer and application of putative channel protein). Over the course of these studies, the number of incorporation attempts for each fraction tested as indicated above was about ten.