Characterization Of Functional nAChR Ensembles

Isotopic ion flux assays are a proven and classical (over 25 years of use) means to characterize function of voltage- or ligand-gated ion channels in clonal cell lines.3536 Ion flux assays complement, and in many ways offer advantages over, more tedious electrophysiological analyses of channel function. Ion flux assays adapted for stopped-flow studies like those used in enzymology give temporal resolution comparable to that for the fastest electrophysiological studies and are fully suitable for intricate study of nAChR functional kinetics.37 Membrane vesicles very rich in nAChR or other channels of interest and resistant to fluid pressures attained in stopped-flow studies, but not to whole cells, are preparations of choice for such studies. Nevertheless, ion flux assays using intact cells are ideally suited for high-throughput analyses of nAChR function using very simple techniques and common instrumentation. Ion flux assays integrate responses for the ensemble of nAChR from the entire population of cells in a cell culture dish or microwell, typically summed across >108 receptors (>105 cells per 15.5-mm diameter well (24-well tray) with ~103 surface receptors per cell).3839 Cellular nAChR responses can be determined using ion flux assays with a temporal resolution of seconds, and ion flux rates typically remain constant over a period of 45 to 60 seconds extrapolated through zero ion flux at time zero. When bi- or multiphasic kinetics of ion flux have been observed, it has reflected time-dependent inactivation of nAChR rather than exhaustion of accessible radiotracer.

Agonist dose-response profiles for test ligands can be obtained from studies of cells plated at equal density in wells in a multiwell array each incubated with a different dose of the test ligand. Positive control (total) responses are determined in samples exposed to a maximally efficacious dose of a standard agonist. Nonspecific ion flux is determined as a negative control in samples lacking agonist or containing both agonist and fully-blocking antagonist. Nonspecific ion flux is subtracted from positive control or test sample responses to yield specific ion flux for those samples. Specific ion flux is plotted as a function of concentration of test ligand, and the data are analyzed using nonlinear regression fits to the formula F = Fmax / (1 + (EC50/[L])n), where F is the measured specific ion flux and [L] is the molar ligand concentration, to yield the parameters Fmax as the maximum ion flux, EC50 as the ligand concentration giving one-half of the maximal ion flux response to the standard agonist, and n (> 0) as the Hill coefficient for the process. Efficacy of test ligand can be determined relative to maximal response to standard agonist in positive control samples. Some test ligands may produce maximal responses at high concentrations that are less than the maximum response to the standard agonist. If agonist dose-response curves for these ligands plateau, then they act as partial agonists and their potencies can be estimated by the concentration giving one-half of their maximal effect. If agonist dose-response curves are bell-shaped, then the test ligand may be exhibiting self-inhibition of functional responses and/or inducing desensitization of nAChR at higher concentrations, but its potency also can be estimated as the dose giving one-half of its maximal effect. EC50 values also will differ from the concentration of a test ligand giving one-half of its maximal effect for the super efficacious drug having >100% of standard agonist efficacy. To provide valid measures of agonist activity, dose-response curves should include measurements at agonist concentrations at least ten times higher than the apparent EC50, and responses at those concentrations should not be more than double responses obtained at the apparent EC50.

Antagonist dose-response curves for test ligands can be obtained from studies of samples treated with different doses of the test ligand in the presence of a standard agonist at a constant concentration. Specific ion flux results are plotted as a function of concentration of test ligand, and the data are analyzed using the formula F = Fmax / (1 + (IC50/[L])n), where F is the measured specific ion flux and [L] is the molar ligand concentration, to yield the parameters Fmax as the maximum ion flux in the absence of antagonist, IC50 as the ligand concentration giving a half-maximal inhibition of ion flux response, and n as the Hill coefficient for the process, which is < 0 for an antagonist in this formula. Tentatively, affinity of nAChR for the test ligand can be expressed as IC50 value (see below). Even if a ligand fails to produce blockade to negative control levels of ion flux, its affinity for nAChR can be tentatively expressed as the concentration giving one-half of that ligand's maximal degree of block.

Competitive or noncompetitive mechanisms of functional blockade can be distinguished based on studies of agonist dose-response profiles at zero and fixed antagonist concentrations. These curves shift to the right (to higher observed EC50 values, thus showing that functional block is surmountable) as competitive antagonist concentrations increase, but they shift downward (reflecting diminished agonist apparent efficacy in the face of insurmountable block) without substantially changing observed EC50 values as noncompetitive antagonist concentrations increase. Similarly, competitive antagonist dose-response profiles will shift to the right (giving increases in apparent IC50 values) as agonist concentrations within the maximally efficacious dose range increase, but noncompetitive antagonist dose-response curves will not shift appreciably left or right as agonist concentrations vary within the maximally efficacious range. IC50 values for noncompetitive antagonists are equal to K values (measures of functional nAChR affinity for the ligand; concentration at which there is half-maximal occupancy of nAChR) regardless of agonist concentration used (although agonist concentration should be equal to or greater than its EC50 value for practical reasons). However, determination of Ki values for competitive antagonists requires additional analysis. The competitive antagonist Ki value can be estimated as the concentration of antagonist that produces a doubling in apparent EC50 value for an agonist in an agonist dose-response profile relative to the EC50 value obtained from such a profile in the absence of antagonist. Competitive antagonist Ki values can be determined more precisely from nonlinear regression analysis of agonist dose-response curves and an expression describing receptor occupancy by agonist and competitive antagonist.40

Ion flux assays can also be used to derive additional information. Use dependence of blockade can be evaluated by testing for enhanced antagonism after short pre-treatment with agonist. Insights into voltage sensitivity of functional blockade can be gained by studies of ion flux responses in the presence of extracellular medium containing different concentrations of potassium ion. Extracellular ion substitution experiments (e.g., N-methyl-D-glucamine exchanged for sodium, Ca2+ removal) can give insights into ion selectivity of channels under study. Spontaneous opening of channels can be assessed by comparing levels of ion flux in the absence of agonist to flux in samples treated with antagonist alone.

On balance, ion flux assays can give information comparable and complementary to that obtained from whole cell current and other methods of electrophysiological recording. Single channel analyses remain the purview of electrical recording. Whole cell current recording has advantages in studies of acute desensitization (occurring in seconds or less) and in some studies of very rapidly inactivating channels that are not open long enough to give significant, integrated ion flux signal above background, such as a7-nAChR. Theoretically, agonist dose-response profiles obtained from peak whole cell currents might differ from those obtained at later times in whole cell current records or from integrated ion flux responses if rates of desensitization of a given nAChR subtype differ across agonists. Similarly, dose-response profiles for a given agonist acting at different nAChR subtypes might differ when taken from peak whole cell currents or when derived from integrated ion flux responses if the two nAChR subtypes have different rates of desensitization. However, there is no reason why antagonist dose-response profiles determined at agonist EC50 values should differ when using, for example, peak whole cell current or integrating ion flux assays. In practice, very few studies have made direct comparisons between ion flux and whole cell current results. Experience shows that dose-response profiles differ more for analysis of a given nAChR subtype expressed in different systems (mammalian cell vs. Xenopus oocyte) than when analyzed using ion flux and electrophysiological techniques in the same expression system.

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