Pharmacological characterization of nAChR can be assessed based on specific binding of radiolabeled nicotinic ligands and on competition by unlabeled compounds for specific radioligand binding. Central to this approach is identification of a suitable radioligand acting with reasonable selectivity (binding with much lower affinity to other nAChR subtypes) or specificity (showing no binding to other nAChR subtypes) at a given nAChR subtype(s). Most radioligands for nAChR are agonists (e.g., 3H-labeled epibatidine, nicotine, acetylcholine, or cytisine) or competitive antagonists (e.g., 125I-labeled a-bungarotoxin or a-cobratoxin; 3H-labeled methyllycaconitine) interacting at overlapping sites on the extracellular face of nAChR. However, high affinity radioligands targeting the channel domain (e.g., 3H-labeled histrionicotoxin or phencyclidine) have been used in studies of preparations highly enriched in nAChR to characterize binding sites for noncompetitive functional antagonists. Radioligand binding studies can be used to make predictions about, or to help confirm, sites of action of functionally potent compounds. For example, ligands acting as competitive antagonists or as agonists in functional assays should also inhibit binding of radiolabeled agonists or competitive antagonists to nAChR with comparable affinities. By contrast, ligands acting as noncompetitive antagonists in functional assays might act with equal potency as competitive inhibitors for radio-ligands binding in the ion channel, but should display no or lower affinity as inhibitors of radiolabeled agonist or competitive antagonist binding.
Essential to radioligand-based characterization of nAChR is determination of numbers of sites and affinities for specific radioligands. Radioligand binding saturation curves are determined for fixed amounts of intact cells or cellular membrane fragments incubated with different concentrations of radioligand. Positive control (total) binding is determined in samples exposed to radioligand alone. Nonspecific binding is determined as a negative control in samples exposed to radioligand and to an at least 100-fold excess of nonradiolabeled probe. Nonspecific binding is subtracted from total binding to yield specific binding for those samples. Plots of specific binding as a function of radioligand concentration are analyzed using nonlinear regression fits commonly found now in data analysis software packages. The simplest analyses assume achievement of equilibrium conditions (see below) and use the formula B = Bmax / (1 + (KD/[L])n), where B is the observed amount of binding and [L] is the radioligand concentration to yield parameters Bmax for the number of specific binding sites, KD for the dissociation constant (a measure of affinity of nAChR for the radioligand), and n as the Hill coefficient for radioligand binding (> 0). Analyses can be extended to assess whether or not more than one class of specific binding sites displaying different affinities for the radioligand exists in a particular preparation and how many receptors exist in each class of binding sites.
Many monographs, review articles, or data analysis software packages explain in further detail transforms for analysis of radioligand binding assays and features of appropriate experimental design and interpretation. Only some of the more salient points will be summarized here. Binding saturation curves should be conducted under conditions where fixed receptor concentration in assay mixtures is less than the KD for the radioligand under use and less than the radioligand concentration. The highest amount of bound radioligand in a saturation assay should be less than 10% of the free radioligand in that sample. This is particularly critical in binding studies using 3H-epibatidine, which can have KD values for binding to some nAChR subtypes of 10 pM or less. Such a high affinity for 3H-epibatidine means that increased reaction volumes are required to lower receptor concentration while maintaining enough receptor to give significant levels of radioligand binding. The highest radioligand concentration in a saturation curve should be ten times higher than KD to help ensure approach to Bmax values. Kinetics of ligand binding should be assessed in concert with saturation binding analyses. Association rate constants (kon) for radioligand binding are typically diffusion controlled yielding values of ~108/mol • min, but slower rates of association are observed for some of the more bulky radioligands, such as 125I-labeled a-bungarotoxin. Observed association rates constants (konobs) are related to true kon by the formula konobs = koff + kon [L], where koff is the dissociation rate constant and [L] is the concentration of radioligand used. There are several ways to determine koff that will not be discussed in detail here. However, KD values for radioligand binding obtained from saturation curves should be consistent with kinetic determinations because of the relationship KD = koff/kon. Moreover, if koff values are greater than 1/min, then dissociation is too fast for most means of sample processing to give reliable data. KD values >10 nM are likely to result. If koff is less than 0.001/min, then half times for dissociation (0.693/koff) will be ~12 hours or more, and KD values <1 pM would be expected. This also means that saturation analyses are complicated, because true equilibrium conditions are not achieved unless samples are incubated for periods equal to four- to five times the half time for radioligand dissociation, and receptor concentrations will need to be lowered in reaction mixtures as discussed previously.
Radioligand binding studies can also be used to derive information about non-radiolabeled ligand interactions at nAChR. Dose-response profiles for test ligands can be obtained from studies of intact cell or cell membrane fractions incubated with different doses of test ligand (preferably covering at least six orders of magnitude) and a constant concentration of radioligand. Positive control (total) and negative control (nonspecific) binding are defined as for saturation curves but at the single concentration of radioligand used. Nonspecific binding is subtracted from positive control or test samples to yield specific binding for those samples. Whether or not equilibrium conditions are achieved,45 formulas describing the competition process have the general structure B = B0 / (1 + (IC50/[L])n), where B is the observed amount of binding, [L] is the test ligand concentration, and B0 is the number of specific binding sites in the absence of competitor, to yield the parameters IC50 as the ligand concentration giving half-maximal blockade of radioligand binding and n as the Hill coefficient for the process (< 0). Affinity of test ligand for nAChR can be expressed tentatively in terms of IC50 value (the test ligand dose giving one-half of the binding in the positive control sample). However, some ligands may not occupy all nAChR interacting with the radioligand and/or will not inhibit more than 50% of radioligand binding. Apparent affinities for the affected subset of nAChR are then based on the test ligand concentration giving one-half of its maximal extent of inhibition of radioligand binding. Ki values (the inhibition constant or the concentration of ligand giving half-maximal occupancy of nAChR) are calculated from the Cheng-Prusoff conversion, Ki = IC50/(1+[L]/KD), where [L] is the concentration of radioligand and KD is its dissociation constant determined kinetically and/or by saturation analysis.
In principle, competitive or noncompetitive mechanisms of inhibition can be distinguished based on studies of radioligand saturation curves at zero and fixed competitor concentrations or on studies of competitor dose-response profiles at different concentrations of radioligand as described above for ion flux analyses. However, these types of studies are often confounded because ligand binding may induce conformational changes in receptors and because binding sites for ligands are not always totally and exclusively overlapping. Moreover, very attractive allosteric models of nAChR hold that binding of a particular ligand stabilizes specific states of nAChR,46 perhaps making those states refractory to binding of other ligands, and giving experimental results indistinguishable from predictions based on noncompetitive mechanisms of radioligand binding block.
For 125I-labeled a-bungarotoxin binding assays conducted in the laboratory, radiolabeled toxin stocks are supplemented with 1 mg/ml of bovine serum albumin, and reaction mixtures contain at least 0.1 mg/ml of bovine serum albumin. This precaution helps to prevent adsorption of radiotoxin to stock or reaction tubes and slows irradiation decomposition of probe. Stock samples are maintained in aqueous solution at -20oC. Nonspecific binding is defined in samples containing 2 |M unlabeled a-bungarotoxin. 3H-labeled agonists are maintained at -20oC in solvents suggested by the manufacturer. For 3H-labeled epibatidine binding assays, we have been able to define nonspecific binding using samples containing 100 | M nicotine in Ringer's buffers that do not require supplementation with bovine serum albumin.
1.5.1 Intact Cells — Suspension and In Situ
Radioligand binding assays of nAChR on the cell surface require use of intact cells assayed either while in suspension or seeded on assay plates.41, 47 To initiate binding assays using plated cells in situ, cell culture growth medium is aspirated. Cells typically prepared on multiwell trays, such as for ion flux assays, are rinsed and equilibrated in ice-cold Ringer's buffer (150 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 1.3 mM MgCl2, 33 mM Tris, pH 7.4, ~310 milliosmolar). The final rinse buffer is aspirated and replaced with assay (Ringer's) buffer containing the radioligand with or without unlabeled homologous ligand or test ligands of choice and at the appropriate single concentration or range of concentrations. Reaction volumes are tailored to the size of the cell culture plate or well. After a prescribed period, the incubation mixture is removed by aspiration. Samples are then rinsed two to three times in ice-cold, fresh Ringer's buffer over a period of minutes (either by sequential aspiration and rinse buffer addition or by application of the flip-plate technique). Rinsed cells are dissolved in 0.01 N NaOH, 0.1% sodium dodecyl sulfate. Dissolved samples are then transferred to appropriate vessels for y-counting of 125I-labeled radioligand bound or to scintillation vials to which liquid scintillation fluid is added for quan-titation of 3H-labeled radioligands.
Cells for radioligand binding assays in suspension are prepared first by aspirating medium from large plates (100-mm diameter in this example) seeded with cells, rinsing cells with ice-cold Ringer's buffer, and adding ~1 ml of ice-cold Ringer's buffer to each dish. Cells are then gently harvested mechanically by scraping the dish bottom with an angled rubber or polypropylene policeman and collecting the cells dislodged by pipette-delivered streams of tangentially applied buffer as a suspension. Cells are gently centrifuged for 3 to 5 min at 500 g, excess buffer is withdrawn, the supernatant is discarded, and the cell pellet is suspended again in Ringer's buffer. The sample is centrifuged, the supernatant withdrawn and discarded, and the pelleted cells are resuspended in fresh Ringer's buffer to a density suitable for the intended assay. Sample aliquots placed in centrifuge tubes (16 mm x 100 mm polycarbonate) are supplemented with radioligand and/or unlabeled ligands of choice and incubated for a prescribed period. Sample volumes are typically 200 | l for 125I-labeled a-bungarotoxin binding assays and 600 |l for 3H-epibatidine binding assays. To end the reaction, samples are diluted in 3 to 4 ml of ice-cold Ringer's buffer, and centrifuged only for the time it takes to accelerate to 5000 rpm. Super-natants are aspirated and discarded, and cell pellets are rapidly but gently re-suspended again in 3 to 4 ml of buffer and centrifuged. The process is repeated for a third cycle of dilute suspension and centrifugation, and the cell pellets are dissolved in 0.01 N NaOH, 0.1% sodium dodecyl sulfate before being processed to quantitate bound radioligand as described previously. Because longer times are needed to process samples using the cell suspension protocol, it is recommended that binding studies using radioligands with fast dissociation rates follow the protocol for intact cells in situ. However, the cell suspension protocol is more economical in terms of reaction volumes and quantities of reagents used.
125I-labeled a-bungarotoxin, which has KD values of ~1 nM for a1*-nAChR or a7-nAChR, can be used in assays with intact cells directly to quantify those nAChR subtypes on the cell surface. In principle, it is also possible to use assays with intact cells to quantify surface and intracellular binding sites for 3H-labeled epibatidine, including those on a4p2-nAChR (typical KD of ~10 pM), a304*-nAChR (typical KD of ~100 pM) or a7-nAChR (typical KD of ~1 nM). Otherwise equivalent samples containing 3H-epibatidine only or 3H-epibatidine plus 10 | M nicotine are used to define total and nonspecific binding, respectively, to cell surface plus intracellular pools of nAChR. Our studies have shown that levels of specific 3H-epibatidine binding to intact cells are indistinguishable from levels of binding to membrane fractions from the same number of cells, showing access of 3H-epibatidine and nicotine to all nAChR pools, even in unbroken cells. Theoretically, positively charged and relatively membrane-impermeant nicotinic ligands such as carbamylcholine should not enter the cell and block intracellular nAChR. Therefore, samples containing 3H-epibatidine plus carbamylcholine should display 3H-epiba-tidine binding to intracellular and nonspecific sites only, allowing specific 3H-epibatidine binding to cell surface sites to be calculated. However, it is recommended that full carbamylcholine competition dose-response profiles be obtained using both intact cell and membrane preparations (see below). This will help determine whether there is any movement of carbamylcholine at higher concentrations into cells. If biphasic competition profiles are obtained when using intact cells, then this indicates that the fraction of binding sites corresponding to surface receptors is blocked by carbamylcholine with high affinity at lower concentrations. The second phase of such a competition profile would indicate that carbamylcholine is entering the cell at higher concentrations and gaining access to intracellular receptors, which would appear to be blocked by carbamylcholine with lower affinity. At a minimum, these studies would help identify a concentration of carbamylcholine adequate to block only surface receptors.
Assays using intact cells should involve incubation at 0 to 4oC to prevent internalization of surface nAChR complexed with radioligand; assay conditions should be chosen to ensure that ligand and receptor concentrations and incubation periods are appropriate to achieve binding equilibrium at these lower temperatures.
1.5.2 Membranes and Detergent-Solubilized Preparations
Preparation of membrane fragments for radioligand binding assays begins with medium removal, rinsing, and mechanical harvesting of cells as described previously. Cell suspensions are gently centrifuged for 3 to 5 min at 500 g, and the supernatant is withdrawn and discarded. The cell pellet is suspended again either in ice-cold, hypo-osmotic 5 mM Tris, pH 7.4 (to help ensure swelling of cells and maximal yield of membranes from small diameter cells such as PC12 and SH-SY5Y) or in ice-cold Ringer's buffer. Samples are subjected to homogenization for 45 seconds using a Polytron at setting 65, using a probe tip, suspension volume, and vessel size to minimize foaming of the suspension. The homogenate is transferred to centrifuge tubes (16 mm x 100 mm polycarbonate) and sedimented at ~40,000 g for 10 minutes. The supernatant is withdrawn and discarded, and the membrane pellet is suspended in fresh Ringer's buffer supplemented with 0.4 mg/ml of sodium azide to a density suitable for the intended assay. If obtained by homogenization in hypotonic buffer, the sample is centrifuged and resuspended in fresh Ringer's buffer one additional time. Brief sonication can be used at this point to aid in obtaining a uniform suspension of membranes. It has been found that resuspension in Ringer's buffer supplemented with 0.4 mg/ml sodium azide allows preservation of membranes in sealed tubes maintained at 4oC for many months without loss of 125I-labeled a-bungarotoxin or 3H-epibatidine binding capacity. Cell pellets can be frozen and stored at -80oC — as can tissues (brain, muscle) — and still yield membrane preparations with preserved nAChR radioligand binding sites. However, nAChR radioligand binding sites are not well preserved by frozen storage of membrane preparations. Membrane fractions suspended in hypo-osmotic buffers (e.g., 25 mM Tris, pH 7.4) have been used by others in nAChR radioligand binding assays, but experience suggests that nonspecific binding is increased substantially under these conditions compared to assays done in Ringer's or other physiological, extracellular salt solutions.
For centrifugation-based assays, membrane sample aliquots, unlabeled ligands of choice, and radioligand are placed in centrifuge tubes (16 mm x 100 mm polycarbonate). Reaction mixtures are gently flicked to ensure mixing after each addition, and reaction tubes are placed on an orbital shaker throughout incubation for the prescribed period. To end the reaction, samples are diluted in 3 to 4 ml of ice-cold Ringer's buffer supplemented with 0.1% bovine serum albumin and cen-trifuged at ~40,000 g for 10 minutes. Supernatants are aspirated and discarded, and membrane pellets are resuspended in 3 to 4 ml of buffer and centrifuged. Resuspension is most efficient if ~250 |l of buffer is added to the tube, allowing the sample to be blended to a fine paste during vigorous vortex mixing, before the bulk of the buffer is added to dilute the suspension. The process is repeated for a third cycle of dilute suspension and centrifugation, and the final cell pellets are dissolved in 0.01 N NaOH, 0.1% sodium dodecyl sulfate before being processed to quantitate bound radioligand as described previously. These assays can be used with radioli-gands that have slower dissociation constants (e.g., 125I-labeled a-bungarotoxin), but they would underestimate numbers of nAChR if assayed with quickly dissociating radioligands. Centrifugation assays of 125I-labeled a-bungarotoxin binding give lower nonspecific binding levels (hence, more resolution) than most filtration assays (but, see below).
For filtration-based assays, sample aliquots are prepared as for centrifugation-based assays but in borosilicate glass tubes (typically 12 mm x 75 mm). Also, Whatman GF/C filters are presoaked in ~0.1 mg/ml of polyethyleneimine before being rinsed with 3 ml of Ringer's buffer just prior to application of reaction samples. After a prescribed period of incubation with orbital shaking, reaction samples of 200 to 600 |l volume are diluted in 3 ml of ice-cold Ringer's buffer. Reaction samples of 6 ml volume can be processed directly. Each diluted suspension is applied to a polyethyleneimine-coated filter. Vacuum is then applied to draw buffer and unbound radioligand through the filter. Three to four ml of Ringer's buffer is added again to each reaction tube, and the contents are transferred to the filter under vacuum, preferably before the filter has dried. The rinse process is repeated twice; 25-mm diameter filter disks or filter pads used in semiautomated sample processors capture comparable quantities of membrane sites. If discs have been used to capture 125I-labeled radioligand binding sites, they can be inserted into test tubes for y counting immediately. Tests should be conducted to ensure that y counting well geometry and sample placement in the counting tube are compatible with maximal detection of isotope. If discs have been used to capture 3H-labeled radioligand binding sites, then they should be dried, placed in vials containing liquid scintillation medium, and left on a shaker overnight to ensure suspension of radiolabel before initiating liquid scintillation counting. Uniformity in efficiency of isotope detection across samples should be ascertained using a liquid scintillation counting internal standard. This is the method of choice for 3H-labeled nAChR agonist binding assays. GF/C filtration-based assays for 125I-labeled a-bungarotoxin binding can be done with greater ease and reproducibility than centrifugation-based assays and with comparable resolution if Ringer's buffer supplemented with 0.1% bovine serum albumin is used to rinse the polyethyleneimine-coated filters prior to and after sample application, as well as in reaction mixtures.
If levels of purity of nAChR allow, ion exchange techniques can be used to capture anionic nAChR-radioligand complexes. The version of the Whatman DE81 disc assay developed by Schmidt and Raftery48 involves application of reaction mixtures to the center of dry, 25-mm diameter DE81 discs placed in ~40-mm diameter, round-bottom wells of a camper's egg carton. Three to four ml of wash solution (50 mM NaCl, 5 mM NaPO4, 0.1% Triton X-100, pH 7.4) are added, and wash solution is removed after 2 to 3 min by aspiration, taking care to flip the filter disc over in the well. This process is repeated twice before discs are processed for determination of bound radioligand as for vacuum-filtered samples.
Specific 3H-epibatidine binding to nonionic detergent-solubilized nAChR (e.g., used in immunoprecipitation assays or to quantitate nAChR fractionated on density gradients) is quantified using GF/C filtration to resolve free 3H-epibatidine from nAChR and bound 3H-epibatidine after the latter have been reprecipitated by addition to a final concentration of 20% polyethylene glycol-8000.
125I-labeled a-bungarotoxin binding assays are typically done in a 200 |l reaction volume containing 100 |l of membranes and 50 |l of radiotoxin at four times the final concentration. Reaction times are typically one hour at room temperature for competition assays, to set up dissociation studies, and for pre-equilibration saturation analyses. 3H-epibatidine binding competition and kinetics assays are typically done using 600 |l reaction volumes containing 100 |l of membranes and 100 |l of 3H-epibatidine at six times the final concentration. 3H-epibatidine saturation assays are done in 6 ml reaction volumes to ensure adequately low concentration of nAChR for assays in the presence of 1-100 pM radioligand. 3H-epibatidine binding reaction times are typically 1 hour at room temperature, which is usually adequate to achieve equilibrium. For most studies, the sequence of reagent addition is dilution buffer, ligand to define nonspecific binding if needed, unlabeled ligand for competition studies if needed, membrane suspension, and then radioligand. If studies are designed to assess unlabeled ligand competition toward initial rates of radioligand binding, then membrane suspension should be added last. For samples containing unlabeled ligand, it should be added in a volume no less than one tenth of the final reaction volume to ensure that there is no more than a ten-fold dilution from the unlabeled ligand stock solution.
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