The neurokinin A receptor activates calcium and cAMP responses through distinct conformational states.

G protein-coupled receptors are thought to mediate agonist-evoked signal transduction by interconverting between discrete conformational states endowed with different pharmacological and functional properties. In order to address the question of multiple receptor states, we monitored rapid kinetics of fluorescent neurokinin A (NKA) binding to tachykinin NK2 receptors, in parallel with intracellular calcium, using rapid mixing equipment connected to real time fluorescence detection. Cyclic AMP accumulation responses were also monitored. The naturally truncated version of neurokinin A (NKA-(4-10)) binds to the receptor with a single rapid phase and evokes only calcium responses. In contrast, full-length NKA binding exhibits both a rapid phase that correlates with calcium responses and a slow phase that correlates with cAMP accumulation. Furthermore, activators (phorbol esters and forskolin) and inhibitors (Ro 31-8220 and H89) of protein kinase C or A, respectively, exhibit differential effects on NKA binding and associated responses; activated protein kinase C facilitates a switch between calcium and cAMP responses, whereas activation of protein kinase A diminishes cAMP responses. NK2 receptors thus adopt multiple activatable, active, and desensitized conformations with low, intermediate, or high affinities and with distinct signaling specificities.

Allosteric mechanisms in normal and pathological nicotinic acetylcholine receptors.

Recent chemical and advanced structural studies on site-directed and naturally occurring pathological mutants of individual members of the multigene family of nicotinic acetylcholine receptors have yielded structure-function relationships supporting indirect 'allosteric' interactions between the acetylcholine-binding sites and the ion channel in signal transduction.

Desensitization of neuronal nicotinic acetylcholine receptors conferred by N-terminal segments of the beta 2 subunit.

Desensitization is a general property of ligand-gated ion channels. Because of a wide array of available subunit combinations, it generates different time constants for channel closure, thereby modulating the processing of information in the brain. Within the family of neuronal nicotinic acetylcholine receptors (nAChRs), alpha 3 beta 2 and alpha 3 beta 4 receptors display contrasting properties of desensitization. When measured using two-electrode voltage-clamp in Xenopus oocytes, desensitization results in current decreases 2 s after initiation of acetylcholine application by 94% for alpha 3 beta 2 receptors, but only by 6% in the case of alpha 3 beta 4 receptors. Desensitization was analyzed by inserting different portions of the beta2 into the beta 4 subunit. Residues 1--212 of the beta2 subunit were able to confer 78% desensitization in 2 s, while smaller chimeras revealed desensitization in 2 s conferred by residues 1--42 alone to a level of 50%, by residues 72--89 to a level of 74%, and by residues 96--212 to a level of 77%. Some long-term (25 min) effects of desensitization driven by acetylcholine were found to rely partially on the same elements, including an enhancement mediated by residues 1--95 and 96--212 of the beta 2 subunit individually. Our results reveal that desensitization relies independently on diverse portions of the extracellular domain of the beta 2 subunit. Phenotype of alpha 3 beta 4 involves, in contrast, complex structural requirements involving residues dispersed throughout the entire N-terminal domain of the beta 4 subunit.

Structural differences in the two agonist binding sites of the Torpedo nicotinic acetylcholine receptor revealed by time-resolved fluorescence spectroscopy.

The nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata carries two nonequivalent agonist binding sites at the alphadelta and alphagamma subunit interfaces. These sites have been characterized by time-resolved fluorescence with the partial nicotinic agonist dansyl-C(6)-choline (Dnscho). When bound to the detergent-solubilized receptor, the fluorescence lifetime distribution of Dnscho displays a characteristic signature, with four separable components at 0.2, 1.8, 7.2, and 18.3 ns, respectively. Competition experiments with the antagonist d-tubocurarine (dTC), known to bind preferentially to the alphagamma site, result in substantial changes of this signature, associated with a strong decrease in average fluorescence lifetime. Comparisons with two other competitive antagonists, alpha-conotoxin M1 and alpha-bungarotoxin, demonstrate that Dnscho binds with a similar affinity to the two sites but that the microenvironment of the probe is different for each site. Using a two-site binding model together with published equilibrium constants to describe the competitive binding of dTC and Dnscho, we reach a satisfactory description of the changes in fluorescence lifetimes and propose characteristic fluorescence parameters of the probe bound to each type of site. This analysis indicates that Dnscho at the alphadelta site is principally associated with a 8.7 ns lifetime, while it has a 20.2 ns major lifetime at the alphagamma site. Therefore, the observed fluorescence heterogeneity arises in large part from the structural differences of the two binding sites. As a result, this signal can be used to identify the binding preferences of competitive ligands of unknown pharmacology.

Chimeric beta-EF3-alpha hemoglobin (Psi): energetics of subunit interaction and ligand binding.

Among the numerous strategies to design an oxygen carrier, we outline in this work the engineering of a stable homotetrameric hemoglobin, expressed in Escherichia coli. The chimeric globin (Psi) consists of the first 79 residues of human beta globin (corresponding to positions NA1 --> EF3) followed by the final 67 residues of human alpha globin (corresponding to positions EF3 --> HC3). The molecular mass for beta-EF3-alpha (Psi) globin was measured using mass spectrometry to be equal to its theoretical value: 15782 Da. Correct protein folding was assessed by UV/visible and fluorescence spectra. The subunit interaction free energies were estimated by HPLC gel filtration. In the cyanometHb species, the formation of the dimer-tetramer interface is 2 kcal/mol less favorable (Delta G = -7 kcal/mol) than that of Hb A (Delta G = -9 kcal/mol), whereas the dimer-monomer interface is tightly assembled (< -10 kcal/mol) as for the Hb A alpha 1 beta 1 interface. In contrast to Hb A, oxygen binding to Psi Hb is not cooperative. The free energy for binding four oxygen molecules to a Psi homotetramer is slightly increased compared to a Hb A heterotetramer (-28 and -27.5 kcal/4 mol of O2, respectively). The intrinsic O2 affinity of a Psi homodimer is 6-fold higher than that of a homotetramer. The linkage scheme between dimer-tetramer subunit assembly and the noncooperative oxygenation of Psi Hb predicts a stabilization of the tetramer after ligand release. This protein mechanism resembles that of Hb A for which the dimers exhibit a 100-fold higher O2 affinity relative to deoxy tetramers (which are 10(5) times more stable than oxy tetramers). A potent allosteric effector of Hb A, RSR4, binds to Psi Hb tetramers, inducing a decrease of the overall O2 affinity. Since RSR4 interacts specifically with two binding sites of deoxy Hb A, we propose that the chimeric tetramer folding is close to this native structure.

Critical elements determining diversity in agonist binding and desensitization of neuronal nicotinic acetylcholine receptors.

To identify the molecular determinants underlying the pharmacological diversity of neuronal nicotinic acetylcholine receptors, we compared the alpha7 homo-oligomeric and alpha4beta2 hetero-oligomeric receptors. Sets of residues from the regions initially identified within the agonist binding site of the alpha4 subunit were introduced into the alpha7 agonist binding site, carried by the homo-oligomeric alpha7-V201-5HT3 chimera. Introduction of the alpha4 residues 183-191 into alpha7 subunit sequence (chimera C2) selectively increased the apparent affinities for equilibrium binding and for ion channel activation by acetylcholine, resulting in a receptor that no longer displays differences in the responses to acetylcholine and nicotine. Introduction of the alpha4 residues 151-155 (chimera B) produced a approximately 100-fold increase in the apparent affinity for both acetylcholine and nicotine in equilibrium binding measurements. In both cases electrophysiological recordings revealed a much smaller increase (three- to sevenfold) in the apparent affinity for activation, but the concentrations required to desensitize the mutant chimeras parallel the shifts in apparent binding affinity. The data were fitted by a two-state concerted model, and an alteration of the conformational isomerization constant leading to the desensitized state accounts for the chimera B phenotype, whereas alteration of the ligand binding site accounts for the chimera C2 phenotype. Point mutation analysis revealed that several residues in both fragments contribute to the phenotypes, with a critical effect of the G152K and T183N mutations. Transfer of alpha4 amino acids 151-155 and 183-191 into the alpha7-V201-5HT3 chimera thus confers physiological and pharmacological properties typical of the alpha4beta2 receptor.

Identifying the conformational state of bi-liganded haemoglobin.

While most researchers agree on the global features of cooperative ligand binding to haemoglobin (Hb), the internal mechanisms remain open to debate. This is not due to inaccurate measurements, but is rather a consequence of the cooperative ligand binding that decreases the equilibrium populations of the partially liganded states and makes observation of the transitions between these substates more difficult. For example, the equilibrium population of the doubly liganded tetramers is typically less than 5% of the total Hb. As a result many models with widely varying mechanisms may fit the oxygen equilibrium curve, but may not be consistent with observations of other parameters, such as ligand-binding kinetics or subunit association equilibria. The wide range of methods and models has led to divergent conclusions about the properties of specific substates. One notable debate concerns the properties of the doubly liganded forms. The simple two-state model predicts a shift in the allosteric equilibrium based on the number of ligands bound, but not on their distribution within the tetramer. From studies of dimer-tetramer equilibria of various pure and hybrid forms, it was concluded that a tetramer with two ligands bound on the same alpha beta dimer (species 21, an asymmetric hybrid) shows an enhanced tetramer stability, similar to singly liganded Hb, relative to the other three types of doubly liganded tetramers which resemble the triply liganded forms [Ackers et al. (1992). Science 255: 54-63]. The implications of this model and the relevant experiments will be reviewed here.

Single binding versus single channel recordings: a new approach to study ionotropic receptors.

The observation of ligand binding to a single molecule has become feasible with recent developments in laser-based fluorescence microscopy. We have simulated such single ligand-binding events for the nicotinic acetylcholine receptor in order to provide comparisons with single channel events under pulsed agonist conditions. The binding events would be more complex than ionic events due to multiple interconversions between different conformational states at the same degree of ligation. Nevertheless, recording of such events could provide valuable new information concerning the role of ligand binding in stabilizing conformational changes and the degree of functional nonequivalence of the binding sites.

Contributions of individual molecular species to the Hill coefficient for ligand binding by an oligomeric protein.

New insights into the Hill coefficient (n) as a measure of cooperativity are obtained by resolving Y, the fractional ligand binding to an oligomeric protein, into a series of integral nth-order reactions. For identical sites within a single conformational state, the weighted sum of each reaction multiplied by its net order gives a Hill coefficient at Y = 0.5 of n50 = 1.0, indicative of non-cooperative binding. However, the disappearance of unliganded oligomers (S0) reflects the higher-order reactions, with their weighted sum (for a tetramer) leading to a Hill coefficient at S0 = 0.5 of n50* = -1.27. For an oligomer with two conformational states (such as represented by the T and R states in the Monod-Wyman-Changeux model) capable of generating highly cooperative binding, the same nth-order reactions apply, but with different weights. For oxygen binding to hemoglobin, n50 is resolved into three components with net reaction orders of n = -2, 2, and 4 (with weights of 0.067, 0.15, and 0.754 corresponding, respectively, to the contributions of singly, triply and quadruply liganded molecules) to give n50 = 3.18. However, the cooperativity of the "state" function, R' (the normalized fraction of molecules in the R state), as characterized by n50' (the Hill coefficient at R' = 0.5) is distinct from n50. If the T-R equilibrium lies very far in favor of either state, then even when the two states differ widely in their intrinsic affinity for ligand, the lower limit of cooperativity for Y is n50 = 1.0, but the Hill coefficient for R' cannot fall below n50' = 1.27 (for a tetramer). Hence, the lower limit of n50' is equal to the absolute value of n50* describing the disappearance of S0 for an oligomer with a single conformational state.

Paradoxical allosteric effects of competitive inhibitors on neuronal alpha7 nicotinic receptor mutants.

Mutation of the conserved leucine residue, in the second transmembrane domain of the neuronal alpha7 acetylcholine receptor to a threonine (L247T) causes pleiotropic alterations of receptor properties. In this study we examined the effects of competitive inhibitors on the alpha7-L247T physiological responses. While the alpha7 competitive inhibitor dihydro-beta-erythroidine evoked a current comparable to that induced by ACh, other inhibitors such as methyllycaconitine (MLA) and alpha-bungarotoxin (alpha-Bgt) caused a blockade of alpha7-L247T to ACh activation. When applied in the absence of ACh, MLA or alpha-Bgt reduced the cell leakage current, showing that alpha7-L247T displays a significant fraction (10%) of spontaneously open channels. These data can be interpreted in terms of an allosteric model, assuming that the L247T mutant possesses a low isomerization constant L and that MLA and alpha-Bgt stabilize the closed, resting state.

Myasthenic nicotinic receptor mutant interpreted in terms of the allosteric model.

An extended Monod-Wyman-Changeux allosteric-type model is applied to human muscle nicotinic acetylcholine receptors expressed in HEK cells, for both the normal form and the high-affinity human myasthenic mutant, epsilon T264P. The model is based on a concerted transition between the basal (resting) B state and the active (open-channel) A state, with the equilibrium in the absence of ligand determined by the allosteric constant, L0 = [B0]/[A0]. For wild-type receptors the model with L0 = 9 x 10(8) provides a satisfactory representation of published patch-clamp recordings that yields a distribution of open-channel dwell times with a single peak at 0.7 ms. For the epsilon T264P mutant, the model with L0 = 100 accounts for the trimodal distribution reported for open-channel dwell times, with peaks at 0.15, 3.8 and 60 ms that correspond to non-, mono- and bi-liganded receptors, respectively. Possible applications of the allosteric model to other myasthenic mutants are considered.

Allosteric proteins after thirty years: the binding and state functions of the neuronal alpha 7 nicotinic acetylcholine receptors.

A key statement of the 1965 Monod-Wyman-Changeux (MWC) model for allosteric proteins concerns the distinction between the ligand-binding function (Y) and the relevant state function (R). Sequential models predict overlapping behavior of the two functions. In contrast, a straightforward experimental consequence of the MWC model is that for an oligomeric protein the parameters which characterize the two functions should differ significantly. Two situations, where R > Y and the system is hyper-responsive or where R < Y and the system is hypo-responsive, have been encountered. Indeed, the hyper-responsive pattern was first observed for the enzyme aspartate transcarbamoylase, by comparing Y with R monitored by a change in sedimentation. Extensions of the theory to ligand-gated channels led to the suggestion that, on the one hand, hyper-responsive properties also occur with high-affinity mutants. On the other hand, native channels of the acetylcholine neuronal alpha 7 receptor and low-affinity mutants of the glycine receptor can be interpreted in terms of the hypo-responsive pattern. For the ligand-gated channels, whereas R is detected directly by ion flux, ligand binding has rarely been measured and the formation of desensitized states may complicate the analysis. However, stochastic models incorporating both binding and channel opening for single molecules predict differences that should be measurable with new experimental approaches, particularly fluorescence correlation spectroscopy.

MHP1, an essential gene in Saccharomyces cerevisiae required for microtubule function.

The gene for a microtubule-associated protein (MAP), termed MHP1 (MAP-Homologous Protein 1), was isolated from Saccharomyces cerevisiae by expression cloning using antibodies specific for the Drosophila 205K MAP. MHP1 encodes an essential protein of 1,398 amino acids that contains near its COOH-terminal end a sequence homologous to the microtubule-binding domain of MAP2, MAP4, and tau. While total disruptions are lethal, NH2-terminal deletion mutations of MHP1 are viable, and the expression of the COOH-terminal two-thirds of the protein is sufficient for vegetative growth. Nonviable deletion-disruption mutations of MHP1 can be partially complemented by the expression of the Drosophila 205K MAP. Mhp1p binds to microtubules in vitro, and it is the COOH-terminal region containing the tau-homologous motif that mediates microtubule binding. Antibodies directed against a COOH-terminal peptide of Mhp1p decorate cytoplasmic microtubules and mitotic spindles as revealed by immunofluorescence microscopy. The overexpression of an NH2-terminal deletion mutation of MHP1 results in an accumulation of large-budded cells with short spindles and disturbed nuclear migration. In asynchronously growing cells that overexpress MHP1 from a multicopy plasmid, the length and number of cytoplasmic microtubules is increased and the proportion of mitotic cells is decreased, while haploid cells in which the expression of MHP1 has been silenced exhibit few microtubules. These results suggest that MHP1 is essential for the formation and/or stabilization of microtubules.

A kinetic mechanism for nicotinic acetylcholine receptors based on multiple allosteric transitions.

Nicotinic acetylcholine receptors are transmembrane oligomeric proteins that mediate interconversions between open and closed channel states under the control of neurotransmitters. Fast in vitro chemical kinetics and in vivo electrophysiological recordings are consistent with the following multi-step scheme. Upon binding of agonists, receptor molecules in the closed but activatable resting state (the Basal state, B) undergo rapid transitions to states of higher affinities with either open channels (the Active state, A) or closed channels (the initial Inactivatable and fully Desensitized states, I and D). In order to represent the functional properties of such receptors, we have developed a kinetic model that links conformational interconversion rates to agonist binding and extends the general principles of the Monod-Wyman-Changeux model of allosteric transitions. The crucial assumption is that the linkage is controlled by the position of the interconversion transition states on a hypothetical linear reaction coordinate. Application of the model to the peripheral nicotine acetylcholine receptor (nAChR) accounts for the main properties of ligand-gating, including single-channel events, and several new relationships are predicted. Kinetic simulations reveal errors inherent in using the dose-response analysis, but justify its application under defined conditions. The model predicts that (in order to overcome the intrinsic stability of the B state and to produce the appropriate cooperativity) channel activation is driven by an A state with a Kd in the 50 nM range, hence some 140-fold stronger than the apparent affinity of the open state deduced previously. According to the model, recovery from the desensitized states may occur via rapid transit through the A state with minimal channel opening, thus without necessarily undergoing a distinct recovery pathway, as assumed in the standard 'cycle' model. Transitions to the desensitized states by low concentration 'pre-pulses' are predicted to occur without significant channel opening, but equilibrium values of IC50 can be obtained only with long pre-pulse times. Predictions are also made concerning allosteric effectors and their possible role in coincidence detection. In terms of future developments, the analysis presented here provides a physical basis for constructing more biologically realistic models of synaptic modulation that may be applied to artificial neural networks.

An allosteric theory for hemoglobin incorporating asymmetric states to test the putative molecular code for cooperativity.

The two-state (MWC) model for cooperative oxygen binding by tetrameric (alpha2beta2) hemoglobin based on concerted transitions between symmetric states (T and R) is extended to include a third, asymmetric state with one alphabeta dimer possessing high (R-like) oxygen affinity and the other alphabeta possessing low (T-like) oxygen affinity. The asymmetric state is assigned a stability that corresponds to the level reported by Ackers and colleagues in the studies on mixed valence hybrids that led to their proposed "molecular code for cooperativity in hemoglobin." However, this level of stability for the asymmetric intermediates significantly diminishes cooperativity in simulated oxygenation curves, to a degree (Hill n = 2.1) that is no longer compatible with the well-established oxygenation properties of normal ferrous hemoglobin (Hill n approximately 3.0). Therefore, the cyanomet derivatives do not appear to be reliable analogues of intermediate oxygenation states.

The multiple phenotypes of allosteric receptor mutants.

Channel-linked neurotransmitter receptors are membrane-bound heterooligomers made up of distinct, although homologous, subunits. They mediate chemo-electrical signal transduction and its regulation via interconversion between multiple conformations that exhibit distinct pharmacological properties and biological activities. The large diversity of functional properties and the widely pleiotropic phenotypes, which arise from point mutations in their subunits (or from subunit substitutions), are interpreted in terms of an allosteric model that incorporates multiple discrete conformational states. The model predicts that three main categories of phenotypes may result from point mutations, altering selectively one (or more) of the following features: (i) the properties of individual binding sites (K phenotype), (ii) the biological activity of the ion channel (gamma phenotype) of individual conformations, or (iii) the isomerization constants between receptor conformations (L phenotype). Several nicotinic acetylcholine and glycine receptor mutants with complex phenotypes are quantitatively analyzed in terms of the model, and the analogies among phenotypes are discussed.

Chimeric hemoglobin subunits: functional properties of a recombinant beta/alpha hemoglobin.

Our goal was to design a single hemoglobin subunit able to assemble into a stable tetrameric structure with cooperative O2 binding and low oxygen affinity. We have synthesized in E. coli a chimeric beta/alpha globin subunit composed of the first 73 residues of the beta chain and the last 73 residues of the alpha chain. Molecular building indicated that this construction could result in Hb homotetramers possessing the alpha 1 beta 2 interface, responsible for the heme-heme interaction in Hb heterotetramers. The results show that the chimeric subunits assemble into tetramers which bind oxygen reversibly without cooperativity but with an oxygen affinity slightly lower than observed for human Hb. The strong effector RSR 4 lowers the oxygen affinity. Kinetics of CO recombination in the presence of RSR 4 reveal a biphasic bimolecular rebinding. Functional studies suggest that the quaternary structure of the oligomer is intermediary between R-and T-state.