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.

Neurotoxicity of channel mutations in heterologously expressed alpha7-nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChR) composed of chick alpha7 subunits mutated to threonine at amino acid valine-251 in the putative channel-lining M2 domain were expressed heterologously in several neuron-like and non-neuronal mammalian cell lines. Expression of mutant alpha7-nAChR is toxic to neuron-like cells of the human neuroblastoma cell lines SH-SY5Y and IMR-32, but not to several other cell types. Growth in the presence of the alpha7-nAChR antagonist methyllycaconitine (MLA) protects against neurotoxicity, as does gradual downregulation of functional, mutant alpha7-nAChR in surviving transfected SH-SY5Y cells. Relative to wild-type alpha7-nAChR, functional alpha7-nAChR mutants show a higher affinity for agonists, slower rates of desensitization, and sensitivity to dihydro-beta-erythroidine (DHbetaE) as an agonist, but they retain sensitivity to MLA as a competitive antagonist. These findings demonstrate that expression of hyperfunctional, mutant forms of Ca2+-permeable alpha7-nAChR is toxic to neuron-like cells.

Optimal inhibition of X4 HIV isolates by the CXC chemokine stromal cell-derived factor 1 alpha requires interaction with cell surface heparan sulfate proteoglycans.

The chemokine stromal cell-derived factor 1 (SDF-1) is the natural ligand for CXC chemokine receptor 4 (CXCR4). SDF-1 inhibits infection of CD4+ cells by X4 (CXCR4-dependent) human immunodeficiency virus (HIV) strains. We previously showed that SDF-1 alpha interacts specifically with heparin or heparan sulfates (HSs). Herein, we delimited the boundaries of the HS-binding domain located in the first beta-strand of SDF-1 alpha as the critical residues. We also provide evidence that binding to cell surface heparan sulfate proteoglycans (HSPGs) determines the capacity of SDF-1 alpha to prevent the fusogenic activity of HIV-1 X4 isolates in leukocytes. Indeed, SDF-1 alpha mutants lacking the capacity to interact with HSPGs showed a substantially reduced capacity to prevent cell-to-cell fusion mediated by X4 HIV envelope glycoproteins. Moreover, the enzymatic removal of cell surface HS diminishes the HIV-inhibitory capacity of the chemokine to the levels shown by the HS-binding-disabled mutant counterparts. The mechanisms underlying the optimal HIV-inhibitory activity of SDF-1 alpha when attached to HSPGs were investigated. Combining fluorescence resonance energy transfer and laser confocal microscopy, we demonstrate the concomitant binding of SDF-1 alpha to CXCR4 and HSPGs at the cell membrane. Using FRET between a Texas Red-labeled SDF-1 alpha and an enhanced green fluorescent protein-tagged CXCR4, we show that binding of SDF-1 alpha to cell surface HSPGs modifies neither the kinetics of occupancy nor activation in real time of CXCR4 by the chemokine. Moreover, attachment to HSPGs does not modify the potency of the chemokine to promote internalization of CXCR4. Attachment to cellular HSPGs may co-operate in the optimal anti-HIV activity of SDF-1 alpha by increasing the local concentration of the chemokine in the surrounding environment of CXCR4, thus facilitating sustained occupancy and down-regulation of the HIV coreceptor.

Subcellular compartmentalization of activation and desensitization of responses mediated by NK2 neurokinin receptors.

A functional fluorescent neurokinin NK2 receptor was constructed by joining enhanced green fluorescent protein to the amino-terminal end of the rat NK2 receptor and was expressed in human embryonic kidney cells. On cell suspensions, the binding of fluorescent Bodipy-labeled neurokinin A results in a saturatable and reversible decrease of NK2 receptor fluorescence via fluorescence resonance energy transfer. This can be quantified for nM to microM agonist concentrations and monitored in parallel with intracellular calcium responses. On single cells, receptor site occupancy and local agonist concentration can be determined in real time from the decrease in receptor fluorescence. Simultaneous measurement of intracellular calcium responses and agonist binding reveals that partial receptor site occupancy is sufficient to desensitize cellular response to a second agonist application to the same membrane area. Subsequent stimulation of a distal membrane area leads to a second response to agonist, provided that it had not been exposed to agonist during the first application. Together with persistent translocation of fluorescent protein kinase C to the membrane area exposed to agonist, the present data support that not only homologous desensitization but also heterologous desensitization of NK2 receptors is compartmentalized to discrete membrane domains.

Molecular basis of the charge selectivity of nicotinic acetylcholine receptor and related ligand-gated ion channels.

Nicotinic acetylcholine receptors are homo- or heteropentameric proteins belonging to the superfamily of receptor channels including the glycine and GABA-A receptors. Affinity labelling and mutagenesis experiments indicated that the M2 transmembrane segment of each subunit lines the ion channel and is coiled into an alpha-helix. Comparison of the M2 sequence of the cation-selective alpha 7 nicotinic receptor to that of the anion-selective alpha 1 glycine receptor identified amino acids involved in charge selectivity. Mutations of the alpha 7 homo-oligomeric receptor within (or near) M2, namely E237A, V251T and a proline insertion P236' were shown to convert the ionic selectivity of alpha 7 from cationic to anionic. Systematic analysis of each of these three mutations supports the notion that the conversion of ionic selectivity results from a local structural reorganization of the 234-238 loop. The 234-238 coiled loop, previously shown to lie near the narrowest portion of the channel, is thus proposed to contribute directly to the charge selectivity filter. A possible functional analogy with the voltage-gated ion channels and related receptors is discussed.

Functional characterization and potential applications for enhanced green fluorescent protein- and epitope-fused human M1 muscarinic receptors.

Four recombinant human M1 (hM1) muscarinic acetylcholine receptors (mAChRs) combining several modifications were designed and overexpressed in HEK293 cells. Three different fluorescent chimera were obtained through fusion of the receptor N terminus with enhanced green fluorescent protein (EGFP), potential glycosylation sites and a large part of the third intracellular (i3) loop were deleted, a hexahistidine tag sequence was introduced at the receptor C terminus, and, finally, a FLAG epitope was either fused at the receptor N terminus or inserted into its shortened i3 loop. High expression levels and ligand binding properties similar to those of the wild-type hM1 receptor together with confocal microscopy imaging demonstrated that the recombinant proteins were correctly folded and targeted to the plasma membrane, provided that a signal peptide was added to the N-terminal domain of the fusion proteins. Their functional properties were examined through McN-A-343-evoked Ca2+ release. Despite the numerous modifications introduced within the hM1 sequence, all receptors retained nearly normal abilities (EC50 values) to mediate the Ca2+ response, although reduced amplitudes (Emax values) were obtained for the i3-shortened constructs. Owing to the bright intrinsic fluorescence of the EGFP-fused receptors, their detection, quantitation, and visualization as well as the selection of cells with highest expression were straightforward. Moreover, the presence of the different epitopes was confirmed by immunocytochemistry. Altogether, this work demonstrates that these EGFP- and epitope-fused hM1 receptors are valuable tools for further functional, biochemical, and structural studies of muscarinic receptors.

Mutational analysis of the charge selectivity filter of the alpha7 nicotinic acetylcholine receptor.

In the alpha7 nicotinic acetylcholine receptors, we analyze the contribution of mutations E237A and V251T, together with the proline insertion P236', in the conversion of the charge selectivity from cationic to anionic. We show that the triple mutant exhibits spontaneous openings displaying anionic selectivity. Furthermore, at position 251, hydrophilic or even negatively charged residues are compatible with an anionic channel. In contrast, the additional proline yields an anionic channel only when inserted between positions 234 and 237; insertion before 234 yields a cationic channel and after 238 alters the receptor surface expression. The coiled 234-238 loop thus directly contributes to the charge selectivity filter of the alpha7 channel.

Fluorescent muscarinic EGFP-hM1 chimeric receptors: design, ligand binding and functional properties.

We describe the construction, expression and characterization of recombinant proteins comprising the enhanced green fluorescent protein (EGFP) fused to the amino-terminal part of the muscarinic hM1 receptor together or not with an additional hexahistidine tag placed at the C-terminal end of the receptor. Expression of the fluorescent proteins reaches levels identical to those of the wt hM1 receptor, provided that fusion takes place at the very N-terminal end of the receptor. Also correct protein folding and targeting to plasma membrane is obtained upon addition of a signal peptide promoting amino-terminal domain translocation through the membrane. Ligand binding properties of--and activation of the calcium release response by--the fusion proteins are almost identical to those of the wild-type muscarinic receptor, indicating that such fluorescently-labelled receptors are valuable model systems for further functional, biochemical and structural studies.

Ivermectin: a positive allosteric effector of the alpha7 neuronal nicotinic acetylcholine receptor.

We report that preapplication of ivermectin, in the micromolar range, strongly enhances the subsequent acetylcholine-evoked current of the neuronal chick or human alpha7 nicotinic acetylcholine receptors reconstituted in Xenopus laevis oocytes and K-28 cells. This potentiation does not result from nonspecific Cl- currents. The concomitant increase in apparent affinity and cooperativity of the dose-response curve suggest that ivermectin acts as a positive allosteric effector. This interpretation is supported by the observation of an increase in efficiency of a partial agonist associated with the potentiation and by the differential effect of ivermectin on mutants within the M2 channel domain. Ivermectin effects reveal a novel allosteric site for pharmacological agents on neuronal alpha7 nicotinic acetylcholine receptors.

Identification of calcium binding sites that regulate potentiation of a neuronal nicotinic acetylcholine receptor.

The divalent cation calcium potentiates the physiological response of neuronal nicotinic receptors to agonists by enhancing ionic current amplitudes, apparent agonist affinity and cooperativity. Here we show that mutations in several consensus Ca2+ binding sequences from the N-terminal domain of the neuronal alpha 7 nicotinic acetylcholine receptor alter Ca2+ potentiation of the alpha 7-V201-5HT3 chimera. Mutations E18Q or E44Q abolish calcium-enhanced agonist affinity but preserve the calcium increase of plateau current amplitudes and cooperativity. On the other hand, mutations of amino acids belonging to the 12 amino acid canonical domain (alpha 7 161-172) alter all features of potentiation by enhancing (D163, S169), reducing (E161, S165, Y167) or abolishing (E172) calcium effects on ionic current amplitudes and agonist affinity. Introduction of the alpha 7 161-172 domain in the calcium insensitive 5-hydroxytryptamine (5HT3) serotoninergic receptor results in a receptor activated by 5HT and potentiated by calcium. In vitro terbium fluorescence studies with an alpha 7 160-174 peptide further show that mutation E172Q also alters in vitro calcium binding. Data are consistent with the occurrence of distinct categories of regulatory calcium binding sites, among which the highly conserved (alpha 7 161-172) domain may simultaneously contribute to calcium and agonist binding.

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.

Identification of a new component of the agonist binding site of the nicotinic alpha 7 homooligomeric receptor.

Tryptophan 54 of the alpha 7 neuronal nicotinic homooligomeric receptor is homologous to gamma-Trp-55 and delta-Trp-57 of non-alpha subunits of Torpedo receptor labeled by d-tubocurarine. This residue was mutated on the alpha 7-V201-5-hydroxytryptamine (5HT)3 homooligomeric chimera, which displays alpha 7 nicotinic pharmacology, and for which both equilibrium binding studies and electrophysiological recordings could be carried out in parallel. Replacement of Trp-54 by a Phe, Ala, or His causes a progressive decrease both in binding affinity and in responses (EC50 or IC50) for acetylcholine, nicotine, and dihydro-beta-erythroidine, without significant modification in alpha-Bgtx binding. Except for Gln-56, comparatively small effects are observed when the other residues of the 52-58 region are mutated into alanine. These data support the participation of Trp-54 to ligand binding, and provide evidence for a new "complementary component" of the alpha 7 nicotinic binding site, distinct from its three-loop "principal component," and homologous to the "non-alpha component" present on gamma and delta subunits.

Chimaeric nicotinic-serotonergic receptor combines distinct ligand binding and channel specificities.

The neuronal nicotinic alpha 7 (nAChR) and 5-hydroxytryptamine (5HT3) receptors are ligand-gated ion channels with a homologous topological organization and have activation and desensitization reactions in common. Yet these homo-oligomeric receptors differ in the pharmacology of their binding sites for agonists and competitive antagonists, and in their sensitivity to Ca2+ ions. The alpha 7 channel is highly permeable to Ca2+ ions and external Ca2+ ions potentiate, in an allosteric manner, the permeability response to acetylcholine, as shown for other neuronal nAChRs. The 5HT3 channel, in contrast, is not permeable to Ca2+ ions, but blocked by them. To assign these properties to delimited domains of the primary structure, we constructed several recombinant chimaeric alpha 7-5HT3 receptors. We report here that one of the constructs expresses a functional receptor that contains the serotonergic channel still blocked by Ca2+ ions, but is activated by nicotinic ligands and potentiated by external Ca2+ ions.

Mutations at two distinct sites within the channel domain M2 alter calcium permeability of neuronal alpha 7 nicotinic receptor.

The relative permeability for sodium, potassium, and calcium of chicken alpha 7 neuronal nicotinic receptor was investigated by mutagenesis of the channel domain M2. Mutations in the "intermediate ring" of negatively charged residues, located at the cytoplasmic end of M2 (site 1), reduce calcium permeability without significantly modifying other functional properties (activation and desensitization) of the receptor; a similar change of ion selectivity is also noticed when mutations at site 1 are done in the context of a receptor mutant that conducts ions in a desensitized state. Moreover, mutations of two adjacent rings of leucines at the synaptic end of M2 (site 2) have multiple effects. They abolish calcium permeability, increase the apparent affinity for acetylcholine by 10- to 100-fold, augment Hill numbers (up to 4.6-5.0) of acetylcholine dose-response relationships, slow rates of ionic response onset, and lower the extent of desensitization. Mutations at these two topographically distinct sites within M2 selectively alter calcium transport without affecting the relative permeabilities for sodium and potassium.

Stratification of the channel domain in neurotransmitter receptors.

Analyses of the ionic pore of ligand-gated ion channels at the amino acid level reveal a structural and functional stratification of the M2 channel domain. Mutations in the equatorial and outer regions affect channel gating, whereas mutations of other amino acid rings alter ionic permeability or selectivity.

A Raman spectroscopic study of acetylcholine receptor-rich membranes from Torpedo marmorata. Interaction of the receptor with carbamylcholine and (+)-tubocurarine.

Raman spectroscopy is used to determine structural features of alkali-treated subsynaptic membrane fragments from Torpedo marmorata electric organ, rich in native functional AcChR. Distinct vibrations attributable to the membrane proteins and lipids were identified and studied before and after addition of the agonist carbamylcholine and the competitive antagonist (+)-tubocurarine. The protein secondary structure determined by using amide-I polypeptide vibrational analysis, indicates 47% alpha-helices, 25% beta-sheets, 18% turns and 11% undefined structure. The secondary structure of the AcChR molecule was not subject to large modifications upon addition of carbamylcholine. But, the presence of the (+)-tubocurarine leads to detectable changes in the amide-I region which might be interpreted as reflecting different contributions of alpha-helices and turns in the secondary structure. In addition, Raman spectra provide information about the environment of aromatic amino acids (tyrosine and tryptophan), the (C-C) bonds, the CH2 and CH3 groups of aliphatic side chains, as well as the disulfide (S-S) and cystein (C-S) bonds. The tyrosines seem 'exposed' to the aqueous medium. The Raman spectra of the AcChR-carbamylcholine complex suggest 'exposed' tryptophans, while those of the unliganded membrane-bound AcChR or of the receptor with (+)-tubocurarine are shown 'buried'. The disulfide bridges in the AcChR subunits show identical conformation in the absence and presence of carbamylcholine. On the contrary, considerable changes are found in the AcChR-(+)-tubocurarine complex. Carbamylcholine and especially (+)-tubocurarine decrease lipid fluidity.

Stratified organization of the nicotinic acetylcholine receptor channel.

Mutations of leucine 247 within the M2 channel domain of the alpha 7 neuronal nicotinic receptor, confer electrophysiological and pharmacological properties, which allow one of the desensitized states to become conductive. Here we show that, in Xenopus oocytes, the effects of the mutations were preserved when 1,2-bis(2-aminophenoxy)-ethane N,N,N',N'-tetraacetic acid (BAPTA) was injected in the cytoplasm to block Ca(2+)-dependent chloride currents, and that in agreement with the proposed interpretation, the ionic currents do not desensitize and rise slowly, in the time-scale of seconds, upon agonist application. Interestingly, similar effects were observed when the two rings (T244, V251) neighbouring L247 on the alpha-helix, but not the more distant ones (S240, L254/255), were mutated, thus supporting the proposal of a functional stratification of the channel domain.