An allosteric model (chemoreception/gustation/membrane receptors/membrane transition)

Transcription

1 Proc. Nati. Acad. Sci. USA Vol. 77, No. 3, pp , NMarch 1980 Neurobiology Sodium and potassium salt stimulation of taste receptor cells: An allosteric model (chemoreception/gustation/membrane receptors/membrane transition) GREGORY MOOSER Department of Biochemistry, School of Dentistry, University of Southern California, Los Angeles, California Communicated by George A. Olah, December 10, 1979 ABSTRACT Stimulation of taste receptors with sodium chloride, sodium acetate, sodium propionate, and the respective potassium salts gave concentration-response profiles, measured electrophysiologically, which are remarkably consistent with a two-state allosteric mechanism. The allosteric cohstant or equilibrium constant for the transition between the active and inactive receptor states is low, resulting in a condition in which small differences in ion affinities for the two states are sufficient to significantly alter the equilibrium. Receptor activators, such as sodium ion, displaced the equilibrium toward the active receptor state by virtue of a higher affinity for that state, whereas receptor inhibitors, such as acetate and propionate ions, displaced the equilibrium in the opposite direction as a result of a higher affinity for the inactive state. The low allosteric constant increased about 10-fold after treatment with the protein modification reagent dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide, resulting in a marked reduction in the response to sodium chloride and sodium propionate without a significant change in ion affinities. In order to fully resolve the potassium response characteristics, it was necessary to consider both a potassium activation site and a potassium inhibition site. Analysis of the response from sodium chloride/potassium chloride mixtures showed that sodium ion is competitive with potassium binding at the activation site but not the inhibition site. With potassium propionate as the stimulus, the effect of both a receptor activator and a receptor inhibitor was quantitatively consistent with depression of the response below a water baseline level at low stimulus concentrations. Estimation of active and inactive state dissociation constants for each anion and cation permitted accurate prediction of the response magnitude for a range of cation ratios in sodium chloride/potassium chloride mixtures and anion ratios in sodium chloride/sodium propionate mixtures. The association of salty taste with receptor activators and bitter taste with receptor inhibitors may be relevant to the generation of these taste qualities. Membrane events in taste receptor cells have been studied electrophysiologically and biochemically with isolated membrane fractions and artificial membranes (1-4). Investigation of salt stimulation poses particular problems because apparent dissociation constants (0.2 M) are too high for direct binding assays. However, electrophysiologic recordings in rats provide a concentration-dependent steady-state response (5) that, on the basis of membrane modification studies, appears to be dependent on events at the receptor cell membrane (6). In this report, sodium and potassium salt stimulation of taste receptors is analyzed in terms of a two-state allosteric mechanism (7). In order to compensate for the indirect electrophysiologic measurements, a variety of anion and cation combinations are examined to evaluate the consistency of the model with taste receptor activation and to estimate stimulus dissociation constants. Allosteric events have been proposed to account for the characteristics of a number of membrane receptor systems (8-11), partly because these models incorporate both ligand binding and conformational transitions. The sodium and potassium salt stimulation data presented here indicate that allosteric events may be important in taste receptor activation as well. The analysis suggests that a low equilibrium constant for the active-inactive state transition can be considered the foundation of a system that is easily displaced toward activation or inactivation by addition of ions that have only small differences in affinity for the two states. Cations, serving as receptor activators, increase the proportion of active-state receptors, and organic anions, serving as receptor inhibitors, increase the proportion of inactive-state receptors. Specific ion affinities, ratios of dissociation constants for the two states, and ion concentrations dictate the position of the equilibrium and magnitude of the response. The state of the equilibrium may not only be associated with stimulus intensity but may also contribute to neural information associated with taste quality discrimination. MATERIALS AND METHODS Activity Measurements. Summated whole nerve chorda tympani recordings on male Holtzman strain white rats were performed as described (12). The magnitude of the tonic activities was linearly reduced to values relative to 100 for 0.3 M NaCl. Preparations were discarded after a significant change in the response to the standard. Activity was measured after the response to a stimulus was stable for a minimum of 90 sec. Each stimulus concentration was applied 2 to 4 times, although not all concentrations required for an experiment were used in each preparation. The total number of preparations used for each experiment is noted in the figure legends. Response magnitudes are shown in the figures with standard errors unless the error falls within the area of the symbol. Inhibition with dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide (HNB-m2S) was carried out with 35 mm reagent at ph 4.0 for 20 min, using previously reported methods (6). Reference activity to NaCl was obtained prior to inhibition; after inhibition, the percent residual activity was periodically monitored as an indication of the integrity of the preparation. Stimuli. Potassium propionate was prepared from propionic acid and potassium hydroxide and was recrystallized three times Abbreviations: OAc, acetate; OPr, propionate; HNB-m2S, dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide. Dissociation The publication costs of this article were defrayed in part by page constants are identified with a subscript indicating stimulus, S, or inhibitor, I, binding to the active state R or the inactive state T of the charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C solely to indicate receptor; e.g., KS(R) is the dissociation constant for stimulus binding this fact. to receptor in the R state. 1686

2 Neurobiology: Mooser from methanol. All other salts were analytical grade reagents obtained from commercial sources. Stimulus solutions were prepared in distilled water and adjusted to ph 7.0 prior to use Ḋata Analysis. Salt concentration-response profiles were analyzed by nonlinear least-squares regression based on allosteric equations for the Monod, Wyman, and Changeux allosteric model, assuming nonexclusive ligand binding (13). With this mechanism, the active state (R) is in equilibrium with the inactive state (T). Ligands bind to both states and displace the equilibrium toward the state with higher affinity. The expression relating the fraction of receptor units in the R state, RI to stimulus concentration and dissociation constants is given in Eq. 1, - ~(1 +a)p (1 + a)n +L( + ca)n [1] in which a is the normalized stimulus concentration (S/KS(R)), L is the allosteric constant or R ± T equilibrium constant, c is the nonexclusive binding coefficient or ratio of R-state to T-state dissociation constants (KS(R)/KS(T)), and n is the smallest common denominator of stimulus binding sites per receptor unit. The relationship between the observed magnitude of activity and R must take into consideration the fraction of R-state units present in the absence of stimulus. Even when the stimulus concentration is zero, R can be significant, as shown by Eq. 2, ifs=o + [2] in which K approaches zero only when L is large. Thus, the observed fraction of R-state receptors measured relative to a baseline response in the absence of stimulus should be expressed as: - ~~~(1+a)n Robs= (1 + a)n + L(l + ca)n 1 + L Because all data are normalized to a value of 100 for 0.3 M NaCl, it was possible to estimate the relative maximum value of R + T by regression analysis. This was found to be 202 and used in all data analyses in the following form: Observed activity - Baseline activity = Robs = Robs X 202. [4] Sodium and potassium salts of organic acids show reduced levels of stimulation relative to the chloride salts. This characteristic was considered in the context of a two-state mechanism by treating the organic anion as a heterotropic allosteric inhibitor-i.e., the anion interacts independently of the cation with a higher affinity for the inactive compared to the active state. An extension of Eq. 3 that incorporates heterotropic inhibition is given in Eq. 5, Robs- (1 + a) n(1 +,r)m (1 + a)n(l + fl)m + L(1 + ca)n(1 + d13)m Proc. Natl. Acad. Sci. USA 77 (1980) L in which : is the normalized inhibitor concentration (I/KI(R)), d is the nonexclusive binding coefficient for the inhibitor (KI(R)/KI(T)), and m is the number of equivalent sites per receptor that bind inhibitor. Frequently, m is equal to n, but, in a membrane receptor system in which conformational transitions of one section of the membrane may place constraints on transitions in another section, inhibitor and stimulus can bind to different macromolecules with no stoichiometric relationship. In the analyses presented here, m was assigned a value of 1 and n was assigned a value of 2, although either 1 or 2 was found acceptable for both n and m. RESULTS The activity from sodium salts has previously been shown to be highest for NaCl, followed by sodium acetate (NaOAc) and then sodium propionate (NaOPr) (5). To test the consistency of the sodium salt response profiles with the allosteric equations, KS(R) and KS(T) for sodium ion were estimated by nonlinear regression using Eq. 3 and the NaCI profile. The values (±15%) were then used in a regression on NaOAc and NaOPr profiles with the organic anion treated as a heterotropic inhibitor according to Eq. 5. The regression curves in Fig. 1 show that the response depression from organic anions can be rationalized by the model without invoking significant alterations in sodium ion interactions. Because a number of models might be expected to adequately resolve this limited data set, several modifications of stimulus conditions were examined. One such modification involves inhibition by HNB-m2S. Under appropriate conditions, HNB-m2S irreversibly reduces the response from sodium salts. The inhibition follows pseudo-first-order kinetics and plateaus with a finite level of residual activity. NaCl concentration does not affect the rate of inhibition but does alter the magnitude of residual activity (6). These characteristics can be explained in the context of a two-state model if HNB-m2S stabilizes the T state with a resultant increase in the allosteric constant. Measurement of NaCl and NaOPr concentration-response profiles after inhibition by HNB-m2S served to provide data for evaluation of this possibility. Regression analyses of the inhibited responses are shown in Fig. 2, where the parameter values are the same as those used in Fig. 1 (+15%) with the exception that L is increased approximately 10-fold. The data are well resolved, suggesting the HNB-m2S inhibi QC Na(CI, OAc, OPr), M FIG. 1. NaCi, NaOAc, and NaOPr response profiles. Data were obtained from 19 preparations. The curves are nonlinear least-squares regressions based on Eq. 3 for NaCl (0) and Eq. 5 for NaOAc (A) and NaOPr (-). Sodium ion parameter values are constant (:t15%) in all of the regressions.

3 1688 Neurobiology: Mooser 0-g Na(CI, OPr), M FIG. 2. Response profiles from stimulation with NaCl (0) and NaOPr (*) after inhibition with 35 mm HNB-m2S for 20 min at ph 4.0. Data from eight preparations are normalized to 100 for the response from 0.3 M NaCl measured prior to inhibition. Regression curves are based on all of the parameter values (+15%) determined from the data in Fig. 1 except the allosteric constant. tion can be viewed as shown below: L R <±T R'. I k * T' HNB-m2S with L estimated from Fig. 1 as 8.8, L' estimated from Fig. 2 as 91.2, and k estimated from prior experiments (6) as 0.12 M-' sec. If the two-state mechanism is valid, it should be possible to predict the response from mixtures of NaCl and NaOPr. The experiment in Fig. 3 shows response magnitudes from mixtures in which sodium concentration was maintained at 0.75 M while chloride and propionate concentrations were varied conversely from 0 to 0.75 M. When the parameter values determined in Fig. 1 were applied to the data, the fit, shown by the broken line, was poor. A possible explanation for this involves competition between chloride and propionate ions. Consideration of chloride interactions was not required to resolve the NaCl data in Fig. 1 and therefore was not incorporated. Note, however, that if chloride binds to the two states with a nonexclusive X 110 ~~f 0 = [0.751 [0.61 [0.41 [0.2] [01 NaOPr, M [NaCI], M FIG. 3. Response magnitudes from NaCl/NaOPr mixtures. The concentration of sodium was 0.75 M with propionate and chloride concentrations varying conversely from 0 to 0.75 M. Data (0) were obtained from five preparations. ---, Regression based on the parameter values determined from the data in Fig. 1. -, Regression based on parameter values in Fig. 1 with the addition of a nonexclusive binding coefficient of 1.0 and dissociation constant of M for chloride binding at the propionate site. Proc. Natl. Acad. Sci. USA 77 (1980) binding coefficient of 1.0 (KI(R) = KI(T)), there would be no effect on the state of the equilibrium even if the ion binds with high affinity. This is readily demonstrated with Eq. 5, in which all of the inhibitor parameters reduce from the equation when d = 1.0, resulting in independence of the state function from anion concentration and affinity. On the other hand, when added in a mixture, chloride would significantly affect propionate binding if the sites overlap. When the experimental data in Fig. 3 were reassessed using a chloride nonexclusive binding coefficient of 1.0 and dissociation constant of M, the fit, shown by the solid line, was excellent. Stimulation with potassium salts results in a reduced magnitude of activity relative to sodium salts, and, because potassium salts elicit both a salty and bitter sensation in humans (14), they.appear to have more complex taste properties. Their application here served to provide an independent evaluation of the organic anion parameters. In addition, KOPr has an interesting characteristic that must be accounted for by the model. Stimulation with potassium benzoate has previously been shown to depress the level of activity below a water-rinse baseline (4), and the same effect was found with KOPr at low concentrations. Under appropriate conditions, Rob, can be less than zero because, when L is small, a finite fraction of the system is in the active form in the absence of stimulus (Eq. 2). In the sodium salt analyses, the calculated value for L was 8.8, which predicts an unstimulated equilibrium with 10% of the receptor units in the R state. Thus, there exists the potential to drive the equilibrium further in the direction of the inactive state. Concentration-response profiles for KCI, KOAc, and KOPr are shown in Fig. 4, with best-fit curves using a value of 8.8 for L and the organic anion parameters (+15%) determined from the sodium salt analyses. All of the data, including the depression of the response below baseline by KOPr, are very well resolved by the equations. However, it was not possible to identify values of potassium binding constants by using only a single class of sites. Beidler (4) proposed two classes of potassium sites to account for its characteristics, and the data reduction in Fig. 4 incorporates both a low-affinity activation site and a highaffinity inhibitory site. It is important to note that even if acetate and propionate affinities are not restricted to the values obtained with sodium salts, adequate resolution requires two distinct potassium sites. The suggestion of two sites leads to the consideration of competition with sodium activation at one or both sites. The analysis chosen to evaluate this is similar to that used with NaCl and NaOPr mixtures. However, in this experiment the chloride Na(CI, OAc, OPr), M FIG. 4. Response profiles from stimulation with KCl (0), KOAc (-), and KOPr (-). Data were obtained from 18 preparations. The curves represent nonlinear least-squares regressions based on the organic anion parameter values (+15%) determined from the data in Fig. 1.

4 Neurobiology: Mooser Proc. Natl. Acad. Sci. USA 77 (1980) n 0 cc 801 K( 1 1t Kl(R KS(R) [0.51 [0.41 [0.31 [0.21 [0.11 [01 KCI, IV [NaCiI, FIG. 5. Response magnitudes from NaCl/KCl mixtures. The concentration of chloride was constant at 0.5 M with potassium and sodium varying conversely from 0 to 0.5 M. Data (0) were obtained from five preparations. The curve is a regression based on the sodium parameter values determined from the data in Fig. 1 and potassium parameter values determined from the data in Fig. 4. The equation for the curve includes sodium competition at the potassium activation site but not at the potassium inhibition site. IKST n0 JKS(R) iks(r) IKS(RJ<L/ concentration in a NaCl/KCl solution was constant, with the two cations varying conversely from 0 to 0.5 M. By incorporating the sodium parameters from Fig. 1 and the potassium parameters from Fig. 4, the mixture response profile was resolved when the equation predicted sodium-potassium competition at the low-affinity activation site (Fig. 5) but not at the high-affinity inhibitory site or at both sites. The calculated values of the parameters for all of the data are listed in Table 1, and the equilibrium interactions in the two-state model are shown schematically in Fig. 6. The complete potassium scheme is not shown but simply involves an additional inhibition site for potassium. The consistency of the data with the model is emphasized by the small variation in anion and cation dissociation constants measured under different conditions. In all of the analyses, the allosteric constant was not varied, and the dissociation constants for each stimulus and inhibitor binding to the R and T states were maintained within ±15%. Because L is small, the ratio of R-state to T-state dissociation constants can be close to 1 and still significantly affect the state of the equilibrium. Thus, for the two stimuli (sodium and potassium at the low-affinity site), the nonexclusive binding Table 1. FIG. 6. Transitions between the active (circles) and inactive (squares) states of the receptor. Equilibria between active and inactive states with identical occupancy are implied but not shown. An S denotes sodium or potassium binding at the activation site, and an I indicates organic anion binding at an inhibition site. Potassium binding at a distinct inhibition site is not shown. The allosteric constant, dissociation constants, and nonexclusive binding coefficients are listed in Table 1. coefficients are only and 0.189, respectively; for the inhibitory interactions (organic anions and potassium at the high-affinity site), the ratio of dissociation constants is even closer to unity, as noted in Table 1. DISCUSSION The magnitude of the tonic response from taste receptor activation by sodium and potassium salts can be explained by a simple two-state allosteric mechanism. A low allosteric constant and nonexclusive ligand binding account for the response characteristics from a large number of anion and cation combinations. Differential affinity of ions for the active and inactive receptor states causes displacement of the equilibrium toward either receptor activation or inhibition. Dissociation constants and allosteric constant for taste receptor cell stimulation with sodium and potassium salts Dissociation constants* Potassium (site 2)t Allosteric Sodium Potassium (site 1)W KI(R) KI(T) Acetate Propionate constant* Stimulus KS(R) KS(T) KS(R) KS(T) X 104 X 104 KI(R) KI(T) KI(R) KI(T) L NaCl NaOAc NaOPr KCl KOAc KOPr NaCl (HNB-m2S treated) NaOPr (HNB-m2S treated) Average (91.2)1 Nonexclusive binding coefficient* * Dissociation constants are in molar units; the allosteric constant and nonexclusive binding coefficients have no units. t Site 1 is an activation site and site 2 is an inhibition site. After inhibition with HNB-m2S.

5 1690 Neurobiology: Mooser Proc. Natl. Acad. Sci. USA 77 (1980) Reduced activity from salts of organic acids compared to chloride salts results from anion binding with an affinity that is slightly higher in the inactive receptor state. The hydrophobic character of the anion appears to be critical for inhibition because chloride ion, which effectively competes with propionate, does not have a differential affinity for the two states and, therefore, does not cause inhibition. A small change in hydrophobic properties at the anion binding site, coupled to the receptor state transition, is sufficient to account for nonexclusive binding coefficients of 1.52 for acetate and 2.55 for propionate. HNB-m2S inhibition is analogous to anion inhibition with the exception that stabilization of the inactive state is irreversible rather than reversible. A 10-fold increase in the allosteric constant after receptor modification accounts for the large reduction in response to NaCl and NaOPr. Postulating changes in anion or cation affinities is not required to resolve the data, as expected from the previous observation that NaCl concentration has no effect on the rate of HNB-m2S inhibition (6). The use of electrophysiologic data to formulate a model for salt activation involves certain compromises. Currently, there is no method to directly monitor salt binding to taste receptor cells, and weak ion affinities together with a paucity of tissue preclude isolation of salt receptors. For this reason, a functional assay of activation was used. The electrophysiologic response has an initial phasic followed by a tonic component. The tonic response is stable for several minutes and appears to be linearly related to events at the cell membrane. Support for this interpretation comes from the observation that inhibitory chemical modification reagents that react at the cell membrane cause a reduction in tonic activity consistent with a psuedo-first-order decay for several half-lives (6). The phasic component, on the other hand, may be generated at the first-order neuron rather than the receptor (15). Thus, the tonic activity was chosen as an assay of the system at a steady state. This does not imply that the tonic component of the response is the sole source of neural information used to judge stimulus intensity and quality. Taste discrimination can occur in time periods in which the phasic component predominates (16). The validity of the analysis is increased by the consistency of the calculated values of equilibrium parameters measured under a variety of stimulus conditions. Each anion and cation dissociation constant was estimated by using two to five separate profiles with different characteristics. For example, the dissociation constants for sodium ion were measured from profiles of the response from NaCG, NaOAc, and NaOPr as well as with NaCl and NaOPr after HNB-m2S modification. Each of the five sodium ion experiments involves a unique set of response modification conditions, all of which give similar values for R-state and T-state sodium ion dissociation constants. In addition, solution mixtures with a range of anion ratios and a range of cation ratios gave response magnitudes that are consistent with that predicted from the analysis of each stimulus alone. The consistency of the data with the equations, given the variations in stimuli, is strong support for a two-state mechanism. Prior research on taste perception provides a basis to speculate on the psychophysical implications of the salt ion interactions. The taste of NaCl is described as salty; cation substitution with potassium adds a bitter component (14) and anion substitution with organic anions adds a taste component that lies outside the domain of all of the four basic taste qualities but is most closely aligned with bitterness (17). Thus, in this limited survey, saltiness is associated with receptor activators such as sodium ion and bitterness is associated with receptor inhibitors such as organic anions. The characteristics of potassium are consistent with this because the analyses presented here indicate that it has distinct activation and inhibition sites that can be correlated with distinct salty and bitter taste qualities. The effects of these various ions on the receptor state may be relevant to the transduction mechanism involved in generation of taste quality. This research was supported in part by Grant DE from the National Institute of Dental Research. 1. Price, S. & DeSimone, J. A. (1977) Chem. Senses Flavor 2, DeSimone, J. A. & Price, S. (1976) Biophys. J. 16, Krueger, J. M. & Cagan, R. H. (1976) J. Biol. Chem. 251, Beidler, L. M. (1962) Prog. Biophys. Biophys. Chem. 12, Beidler, L. M. (1954) J. Gen. Physiol. 34, Mooser, G. & Lambuth, N. (1977) J. Neurobiol. 8, Monod, J., Wyman, J. & Changeux, J.-P. (1965) J. Mol. Biol. 12, Catterall, W. A. (1977) J. Biol. Chem. 252, Limbird, L. E. & Lefkowitz, R. J. (1976) J. Biol. Chem. 251, DeMeyts, P. (1976) J. Supramol. Struct. 4, Edelstein, S. J. (1972) Biochem. Biophys. Res. Commun. 48, Mooser, G. (1976) J. Neurobiol. 7, Rubin, M. M. & Changeux, J.-P. (1966) J. Mol. Biol. 21, McBurney, D. H. & Shick, T. R. (1971) Percept. Psychophys. 10, Sato, T. (1976) Experientia 32, Halpern, B. P. & Tapper, D. N. (1971) Science 171, Schiffman, S. S. (1980) in Biological and Behavioral Aspects of Salt Intake, eds. Kare, M. R., Fregly, M. H. & Bernard, R. A. (Academic, New York), in press.