Purification of a native nicotinic receptor.

Nicotinic acetylcholine receptors are members of the Cys-loop superfamily of pentameric ligand-gated ion channels. The electric organ of the Torpedo ray is extraordinarily rich in an acetylcholine receptor that is homologous to the human nicotinic receptor found at the neuromuscular junction. Due to this abundant natural source in the fish and the relatively accessible preparation of the neuromuscular junction (compared to a central synapse), this muscle-type receptor and specifically the fish receptors have long been used as the prototype for study of nicotinic receptors. However, an absence of structural detail at high resolution has limited the chemical interpretation of this archetypal nicotinic receptor. One of the main concerns in preparing receptor for high resolution structural analysis was its documented sensitivity to particular detergents and requirements for specific lipids in order to maintain function after reconstitution in a membrane. Here, we present methods for purifying native nicotinic receptor from Torpedo electric tissue that maintains functionality after reconstitution and that is amenable to high resolution structural analysis. The specific developments we describe include detergent exchange during purification, inclusion of specific lipids during purification and for nanodisc reconstitution, and synthesis of a new affinity reagent for rapid isolation of receptors.

Sequential purification and characterization of Torpedo californica nAChR-DC supplemented with CHS for high-resolution crystallization studies.

Over the past 10 years we have been developing a multi-attribute analytical platform that allows for the preparation of milligram amounts of functional, high-pure, and stable Torpedo (muscle-type) nAChR detergent complexes for crystallization purpose. In the present work, we have been able to significantly improve and optimize the purity and yield of nicotinic acetylcholine receptors in detergent complexes (nAChR-DC) without compromising stability and functionality. We implemented new methods in the process, such as analysis and rapid production of samples for future crystallization preparations. Native nAChR was extracted from the electric organ of Torpedo californica using the lipid-like detergent LysoFos Choline 16 (LFC-16), followed by three consecutive steps of chromatography purification. We evaluated the effect of cholesteryl hemisuccinate (CHS) supplementation during the affinity purification steps of nAChR-LFC-16 in terms of receptor secondary structure, stability and functionality. CHS produced significant changes in the degree of ß-secondary structure, these changes compromise the diffusion of the nAChR-LFC-16 in lipid cubic phase. The behavior was reversed by Methyl-ß-Cyclodextrin treatment. Also, CHS decreased acetylcholine evoked currents of Xenopus leavis oocyte injected with nAChR-LFC-16 in a concentration-dependent manner. Methyl-ß-Cyclodextrin treatment do not reverse functionality, however column delipidation produced a functional protein similar to nAChR-LFC-16 without CHS treatment.

d-Amino Acid Substitution of α-Conotoxin RgIA Identifies its Critical Residues and Improves the Enzymatic Stability.

α-Conotoxin RgIA is a selective and potent competitive antagonist of rat α9α10 nicotinic acetylcholine receptors (nAChR), but it is much less potent towards human α9α10 nAChR. Furthermore, RgIA is susceptible to proteolytic degradation due to containing four arginine residues. These disadvantages greatly limit its use for clinical applications. The purpose of this research was to identify critical stereocenters of RgIA and discover more stable analogues, enhancing its bioavailability by using the d-amino acid scan method. The activity of each variant was investigated against rat and human α9α10 nAChRs, which were expressed in Xenopus oocytes. Experimental assays showed that 14 out of 15 analogues had a substantial reduction in potency towards rat α9α10 nAChR. Noticeably, analogue 13 retained full biological activity compared with RgIA. Meanwhile, two other analogues, 14 and 15, of which l-Args were substituted with d-Args, exhibited a significantly increased potency towards human α9α10 nAChR, although these analogues showed decreased activities against rat α9α10 nAChR. Additionally, these three analogues exhibited a high resistance against enzymatic degradation in human serum and simulated intestinal fluid (SIF). Collectively, our findings suggest that a d-amino acid scan is a useful strategy for investigating how the side-chain chirality of amino acids affects the structure and function of peptides and may facilitate the development of more stable analogues to increase therapeutic potential.

Exploring the Metabolism of (+)-[18F]Flubatine in Vitro and in Vivo: LC-MS/MS Aided Identification of Radiometabolites in a Clinical PET Study.

Both (+)-[18F]flubatine and its enantiomer (-)-[18F]flubatine are radioligands for the neuroimaging of α4ß2 nicotinic acetylcholine receptors (nAChRs) by positron emission tomography (PET). In a clinical study in patients with early Alzheimer's disease, (+)-[18F]flubatine ((+)-[18F]1) was examined regarding its metabolic fate, in particular by identification of degradation products detected in plasma and urine. The investigations included an in vivo study of (+)-flubatine ((+)-1) in pigs and structural elucidation of formed metabolites by LC-MS/MS. Incubations of (+)-1 and (+)-[18F]1 with human liver microsomes were performed to generate in vitro metabolites, as well as radiometabolites, which enabled an assignment of their structures by comparison of LC-MS/MS and radio-HPLC data. Plasma and urine samples taken after administration of (+)-[18F]1 in humans were examined by radio-HPLC and, on the basis of results obtained in vitro and in vivo, formed radiometabolites were identified.

Assessment of the functionality and stability of detergent purified nAChR from Torpedo using lipidic matrixes and macroscopic electrophysiology.

In our previous study we examined the functionality and stability of nicotinic acetylcholine receptor (nAChR)-detergent complexes (nAChR-DCs) from affinity-purified Torpedo californica (Tc) using fluorescence recovery after photobleaching (FRAP) in Lipidic Cubic Phase (LCP) and planar lipid bilayer (PLB) recordings for phospholipid and cholesterol like detergents. In the present study we enhanced the functional characterization of nAChR-DCs by recording macroscopic ion channel currents in Xenopus oocytes using the two electrode voltage clamp (TEVC). The use of TEVC allows for the recording of macroscopic currents elicited by agonist activation of nAChR-DCs that assemble in the oocyte plasma membrane. Furthermore, we examined the stability of nAChR-DCs, which is obligatory for the nAChR crystallization, using a 30 day FRAP assay in LCP for each detergent. The present results indicate a marked difference in the fractional fluorescence recovery (ΔFFR) within the same detergent family during the 30 day period assayed. Within the cholesterol analog family, sodium cholate and CHAPSO displayed a minimum ΔFFR and a mobile fraction (MF) over 80%. In contrast, CHAPS and BigCHAP showed a marked decay in both the mobile fraction and diffusion coefficient. nAChR-DCs containing phospholipid analog detergents with an alkylphosphocholine (FC) and lysofoscholine (LFC) of 16 carbon chains (FC-16, LFC-16) were more effective in maintaining a mobile fraction of over 80% compared to their counterparts with shorter acyl chain (C12, C14). The significant differences in macroscopic current amplitudes, activation and desensitization rates among the different nAChR-DCs evaluated in the present study allow to dissect which detergent preserves both, agonist activation and ion channel function. Functionality assays using TEVC demonstrated that LFC16, LFC14, and cholate were the most effective detergents in preserving macroscopic ion channel function, however, the nAChR-cholate complex display a significant delay in the ACh-induce channel activation. In summary, these results suggest that the physical properties of the lipid analog detergents (headgroup and acyl chain length) are the most effective in maintaining both the stability and functionality of the nAChR in the detergent solubilized complex.

Purification and functional characterization of a truncated human α4ß2 nicotinic acetylcholine receptor.

Nicotinic acetylcholine receptors (nAChR) are abundant in the brain and are essential in cognitive function, learning and memory. Previous efforts on α4ß2 nAChR had been focused on functional and pharmacological characterization, where high expression yield is not essential. For structural studies though, large amounts of pure protein is important but heterologous overexpression of membrane proteins can be a burdensome task, especially if high amounts are required. In the current study, a truncated mutant of the human α4ß2 nAChR was designed in order to improve expression and solubility and to obtain material suitable for high resolution structural studies. We showed that the wild type α4ß2 nAChR presented low expression and solubilization yield both of which were improved with the truncated construct. The truncated nAChR showed similar binding profile to the wild type, was purified by a two-step chromatography and isolated in high purity and adequate quantity.

Experimental autoimmune myasthenia gravis in the mouse.

Myasthenia gravis (MG) is a T cell-dependent antibody-mediated autoimmune neuromuscular disease. Antibodies to the nicotinic acetylcholine receptor (AChR) destroy the AChR, thus leading to defective neuromuscular transmission of electrical impulse and to muscle weakness. This unit is a practical guide to the induction and evaluation of experimental autoimmune myasthenia gravis (EAMG) in the mouse, the animal model for MG. Protocols are provided for the extraction and purification of AChR from the electric organs of Torpedo californica, or the electric ray. The purified receptor is used as an immunogen to induce autoimmunity to AChR, thus causing EAMG. The defect in neuromuscular transmission can also be measured quantitatively by electromyography. In addition, EAMG is frequently characterized by the presence of serum antibodies to AChR, which are measured by radioimmunoassay and by a marked antibody-mediated reduction in the number of muscle AChRs. AChR extracted from mouse muscle is used in measuring serum antibody levels and for quantifying muscle AChR content. Another hallmark of the disease is complement and IgG deposits located at the neuromuscular junction, which can be visualized by immunofluorescence techniques.

The nicotinic acetylcholine receptors of zebrafish and an evaluation of pharmacological tools used for their study.

Zebrafish (Danio rerio) have been used to study multiple effects of nicotine, for example on cognition, locomotion, and stress responses, relying on the assumption that pharmacological tools will operate similarly upon molecular substrates in the fish and mammalian systems. We have cloned the zebrafish nicotinic acetylcholine receptor (nAChR) subunits and expressed key nAChR subtypes in Xenopus oocytes including neuronal (α4ß2, α2ß2, α3ß4, and α7) and muscle (α1ß1(b)ɛδ) nAChR. Consistent with studies of mammalian nAChR, nicotine was relatively inactive on muscle-type receptors, having both low potency and efficacy. It had high efficacy but low potency for α7 receptors, and the best potency and good efficacy for α4ß2 receptors. Cytisine, a key lead compound for the development of smoking cessation agents, is a full agonist for both mammalian α7 and α3ß4 receptors, but a full agonist only for the fish α7, with surprisingly low efficacy for α3ß4. The efficacy of cytisine for α4ß2 was somewhat greater than typically reported for mammalian α4ß2. The ganglionic blocker mecamylamine was most potent for blocking α3ß4 receptors, least potent for α7, and roughly equipotent for the muscle receptors and the ß2-containing nAChR. However, the block of ß2-containing receptors was slowly reversible, consistent with effective targeting of these CNS-type receptors in vivo. Three prototypical α7-selective agonists, choline, tropane, and 4OH-GTS-21, were tested, and these agents were observed to activate both fish α7 and α4ß2 nAChR. Our data therefore indicate that while some pharmacological tools used in zebrafish may function as expected, others will not.

[Immunocytochemical identification of synaptotagmin 1, syntaxin 1, N-type Ca(2+)-channel and nicotinic cholinoreceptor in motor neuromuscular junctions of earthworm somatic muscle Lumbricus terrestris].

The somatic muscle of earthworm contains myoneural synapses forming clusters of "synaptic buttons". In these "buttons", the proteins syntaxin 1, synaptotagmin 1 and alpha 1B subunit of the Ca(2+)-channel of N-type were identified. We suppose that "synaptic buttons" contain a limited number of active zones due to their small size (1-2 microm) and the pattern of distribution of proteins of exo-endocytotic cycle. The postsynaptic membrane of cholinetgic synapses contains nicotinic acetylcholine receptors capable to bind alpha-bungarotoxin. The area of location of receptors on postsynaptic membrane is strictly limited to the region of synaptic contact.

RegIIA: an α4/7-conotoxin from the venom of Conus regius that potently blocks α3ß4 nAChRs.

Neuronal nicotinic acetylcholine receptors (nAChRs) play pivotal roles in the central and peripheral nervous systems. They are implicated in disease states such as Parkinson's disease and schizophrenia, as well as addictive processes for nicotine and other drugs of abuse. Modulation of specific nAChRs is essential to understand their role in the CNS. α-Conotoxins, disulfide-constrained peptides isolated from the venom of cone snails, potently inhibit nAChRs. Their selectivity varies markedly depending upon the specific nAChR subtype/α-conotoxin pair under consideration. Thus, α-conotoxins are excellent probes to evaluate the functional roles of nAChRs subtypes. We isolated an α4/7-conotoxin (RegIIA) from the venom of Conus regius. Its sequence was determined by Edman degradation and confirmed by sequencing the cDNA of the protein precursor. RegIIA was synthesized using solid phase methods and native and synthetic RegIIA were functionally tested using two-electrode voltage clamp recording on nAChRs expressed in Xenopus laevis oocytes. RegIIA is among the most potent antagonist of the α3ß4 nAChRs found to date and is also active at α3ß2 and α7 nAChRs. The 3D structure of RegIIA reveals the typical folding of most α4/7-conotoxins. Thus, while structurally related to other α4/7 conotoxins, RegIIA has an exquisite balance of shape, charge, and polarity exposed in its structure to potently block the α3ß4 nAChRs.

Experimental autoimmune myasthenia gravis in the mouse.

Myasthenia gravis (MG) is a T cell-dependent antibody-mediated autoimmune neuromuscular disease. Antibodies to the nicotinic acetylcholine receptor (AChR) destroy the AChR, thus leading to defective neuromuscular transmission of electrical impulse and to muscle weakness. This unit is a practical guide to the induction and evaluation of experimental autoimmune myasthenia gravis (EAMG) in the mouse, the animal model for MG. Protocols are provided for the extraction and purification of AChR from the electric organs of Torpedo californica, or the electric ray. The purified receptor is used as an immunogen to induce autoimmunity to AChR, thus causing EAMG. The defect in neuromuscular transmission can also be measured quantitatively by electromyography. In addition, EAMG is frequently characterized by the presence of serum antibodies to AChR, which are measured by radioimmunoassay and by a marked antibody-mediated reduction in the number of muscle AChRs. AChR extracted from mouse muscle is used in measuring serum antibody levels and for quantifying muscle AChR content. Another hallmark of the disease is complement and IgG deposits located at the neuromuscular junction, which can be visualized by immunofluorescence techniques.

Effects of lipid-analog detergent solubilization on the functionality and lipidic cubic phase mobility of the Torpedo californica nicotinic acetylcholine receptor.

Over the past three decades, the Torpedo californica nicotinic acetylcholine receptor (nAChR) has been one of the most extensively studied membrane protein systems. However, the effects of detergent solubilization on nAChR stability and function are poorly understood. The use of lipid-analog detergents for nAChR solubilization has been shown to preserve receptor stability and functionality. The present study used lipid-analog detergents from phospholipid-analog and cholesterol-analog detergent families for solubilization and affinity purification of the receptor and probed nAChR ion channel function using planar lipid bilayers (PLBs) and stability using analytical size exclusion chromatography (A-SEC) in the detergent-solubilized state. We also examined receptor mobility on the lipidic cubic phase (LCP) by measuring the nAChR mobile fraction and diffusion coefficient through fluorescence recovery after photobleaching (FRAP) experiments using lipid-analog and non-lipid-analog detergents. Our results show that it is possible to isolate stable and functional nAChRs using lipid-analog detergents, with characteristic ion channel currents in PLBs and minimal aggregation as observed in A-SEC. Furthermore, fractional mobility and diffusion coefficient values observed in FRAP experiments were similar to the values observed for these parameters in the recently LCP-crystallized ß(2)-adrenergic receptor. The overall results show that phospholipid-analog detergents with 16 carbon acyl-chains support nAChR stability, functionality and LCP mobility.

Affinity purification of a chimeric nicotinic acetylcholine receptor in the agonist and antagonist bound states.

Nicotinic acetylcholine receptors (nAChRs) form ligand-gated ion channels that mediate fast signal transmission at synapses. These receptors are members of a large family of pentameric ion channels that are of active medical interest. An expression system utilizing a chimerical construct of the N-terminal extracellular ligand binding domain of alpha7 type nAChR and the C-terminal transmembrane portion of 5HT3 type receptor resulted high level of expressions. Two ligand affinity chromatography purification methods for this receptor have been developed. One method relies on the covalent immobilization of a high affinity small molecule alpha7 nAChR agonist, (R)-5-(4-aminophenyl)-N-(quinuclidin-3-yl) furan-2-carboxamide, and the other uses mono biotinylated alpha-bungarotoxin, an antagonist, that forms a quasi-irreversible complex with alpha7 nAChR. Detergent solubilized alpha7/5HT(3) chimeric receptors were selectively retained on the affinity resins and could be eluted with free ligand or biotin. The proteins purified by both methods were characterized by gel electrophoresis, mass spectra, amino acid composition analysis, and N-terminal sequence determination. These analyses confirmed the isolation of a mature alpha7/5HT(3) receptor with the signal peptide removed. These results suggest a scalable path forward to generate multi-milligram amounts of purified complexes for additional studies including protein crystallization.

Phospholipase C activity affinity purifies with the Torpedo nicotinic acetylcholine receptor.

Nicotinic acetylcholine receptors mediate fast synaptic transmission by fluxing ions across the membrane in response to neurotransmitter binding. We show here that during affinity purification of the nicotinic acetylcholine receptor from Torpedo, phosphatidic acid, but not other anionic or zwitterionic phospholipids, is hydrolyzed to diacylglycerol. The phospholipase C activity elutes with the acetylcholine receptor and is inhibited by a lipid phosphate phosphohydrolase inhibitor, sodium vanadate, but not a phosphatidate phosphohydrolase inhibitor, N-ethylmaleimide. Further, the hydrolysis product of phosphatidic acid, diacylglycerol, enhances the functional capabilities of the acetylcholine receptor in the presence of anionic lipids. We conclude that a phospholipase C activity, which appears to be specific for phosphatidic acid, is associated with the nicotinic acetylcholine receptor. The acetylcholine receptor may directly or indirectly influence lipid metabolism in a manner that enhances its own function.

Interaction of lipids and ligands with nicotinic acetylcholine receptor vesicles assessed by electron paramagnetic resonance spectroscopy.

Electron paramagnetic resonance (EPR) spectroscopy is a powerful technique that permits the study of membrane-embedded proteins in its lipid environment by assessing the interaction of spin labels with the protein in its natural environment (i.e., native membranes) or in reconstituted systems prepared with exogenous lipid species. Nicotinic acetylcholine receptors (AChRs) contain a large surface in intimate contact with the lipid membrane. AChRs, members of the Cys-loop receptor superfamily, have essential functional roles in the nervous system and its malfunctioning has been considered as the origin of several neurological diseases including Alzheimer's disease, drug addiction, depression, and schizophrenia. In this regard, these receptors have been extensively studied as therapeutic targets for the action of several drugs. The majority of the marketed medications bind to the neurotransmitter sites, the so-called agonists. However, several drugs, some of them still in clinical trials, interact with non-competitive antagonist (NCA) binding sites. A potential location for these binding sites is the proper ion channel, blocking ion flux and thus, inhibiting membrane depolarization. However, several NCAs also bind to the lipid-protein interface, modulating the AChR functional properties. The best known examples of these NCAs are local and general anesthetics. Several endogenous molecules such as free fatty acids and neurosteroids also bind to the lipid-protein interface, probably mediating important physiological functions. Phospholipids, natural components of lipid membranes interacting with the AChR, are also essential to maintain the structural and functional properties of the AChR. EPR studies showed that local anesthetics bind to the lipid-protein interface by essentially the same dynamic mechanisms found in lipids, and that local and general anesthetics preferably decrease the phospholipid but not the fatty acid interactions with the AChR. This is consistent with the existence of annular and non-annular lipid domains on the AChR.

Probing the structure of the affinity-purified and lipid-reconstituted torpedo nicotinic acetylcholine receptor.

The Torpedo nicotinic acetylcholine receptor (nAChR) is the only member of the Cys-loop superfamily of ligand-gated ion channels (LGICs) that is available in high abundance in a native membrane preparation. To study the structure of the other LGICs using biochemical and biophysical techniques, detergent solubilization, purification, and lipid reconstitution are usually required. To assess the effects of purification on receptor structure, we used the hydrophobic photoreactive probe 3-trifluoromethyl-3-(m-[(125)I]iodophenyl)diazirine ([(125)I]TID) to compare the state-dependent photolabeling of the Torpedo nAChR before and after purification and reincorporation into lipid. For the purified nAChR, the agonist-sensitive photolabeling within the M2 ion channel domain of positions M2-6, M2-9, and M2-13, the agonist-enhanced labeling of deltaThr274 (deltaM2-18) within the delta subunit helix bundle, and the labeling at the lipid-protein interface (alphaMu4) were the same as for the nAChR in native membranes. However, addition of agonist did not enhance [(125)I]TID photolabeling of deltaIle288 within the deltaM2-M3 loop. These results indicate that after purification and reconstitution of the Torpedo nAChR, the difference in structure between the resting and desensitized states within the M2 ion channel domain was preserved, but not the agonist-dependent change of structure of the deltaM2-M3 loop. To further characterize the pharmacology of [(125)I]TID binding sites in the nAChR in the desensitized state, we examined the effect of phencyclidine (PCP) on [(125)I]TID photolabeling. PCP inhibited [(125)I]TID labeling of amino acids at the cytoplasmic end of the ion channel (M2-2 and M2-6) while potentiating labeling at M2-9 and M2-13 and allosterically modulating the labeling of amino acids within the delta subunit helix bundle.

Lipid composition alters drug action at the nicotinic acetylcholine receptor.

We tested the hypothesis that membrane lipid composition influences drug action at membrane proteins by studying local anesthetic action at the nicotinic acetylcholine receptor (nAChR). Infrared difference spectra show that concentrations of tetracaine consistent with binding to the ion channel (<50 microM) stabilize a resting-like state when the nAChR is reconstituted into phosphatidylcholine membranes containing the anionic lipid, phosphatidic acid, but have no effect on the nAChR reconstituted into membranes lacking phosphatidic acid, either in the presence or absence of cholesterol. Concentrations of tetracaine above 200 microM lead to neurotransmitter site binding in all membranes. In the presence of phosphatidic acid, cholesterol, or both, neurotransmitter site binding leads to the formation of quaternary amine-aromatic interactions between tetracaine and binding site tyrosine/tryptophan residues and the stabilization of a desensitized state. One interpretation suggested by lipid partitioning studies is that phosphatidic acid enhances tetracaine action at the channel pore by increasing the partitioning of tetracaine into the lipid bilayer, thereby enhancing access to the transmembrane pore. However, subtle membrane-dependent variations in the vibrations of tyrosine and tryptophan residues, and agonist analog binding studies indicate that the structures of the agonist-bound neurotransmitter sites of the nAChR in membranes lacking both phosphatidic acid and cholesterol differ from the structures of the agonist-desensitized neurotransmitter sites in the presence of both lipids. Lipid action at the nAChR thus involves more than a simple modulation of the equilibrium between resting and desensitized states.

Cloning and characterization of the hamster and guinea pig nicotinic acid receptors.

In this study, we present the identification and characterization of hamster and guinea pig nicotinic acid receptors. The hamster receptor shares approximately 80-90% identity with the nucleotide and amino acid sequences of human, mouse, and rat receptors. The guinea pig receptor shares 76-80% identity with the nucleotide and amino acid sequences of these other species. [(3)H]nicotinic acid binding affinity at guinea pig and hamster receptors is similar to that in human (dissociation constant = 121 nM for guinea pig, 72 nM for hamster, and 74 nM for human), as are potencies of nicotinic acid analogs in competition binding studies. Inhibition of forskolin-stimulated cAMP production by nicotinic acid and related analogs is also similar to the activity in the human receptor. Analysis of mRNA tissue distribution for the hamster and guinea pig nicotinic acid receptors shows expression across a number of tissues, with higher expression in adipose, lung, skeletal muscle, spleen, testis, and ovary.

Nicotinic alpha5 subunit deletion locally reduces high-affinity agonist activation without altering nicotinic receptor numbers.

Neuronal nicotinic acetylcholine receptor subunit alpha5 mRNA is widely expressed in the CNS. An alpha5 gene polymorphism has been implicated in behavioral differences between mouse strains, and alpha5-null mutation induces profound changes in mouse acute responses to nicotine. In this study, we have examined the distribution and prevalence of alpha5* nicotinic acetylcholine receptor in mouse brain, and quantified the effects of alpha5-null mutation on pre-synaptic nicotinic acetylcholine receptor function (measured using synaptosomal (86)Rb(+) efflux) and overall [(125)I]epibatidine binding site expression. alpha5* nicotinic acetylcholine receptor expression was found in nine of fifteen regions examined, although < 20% of the total nicotinic acetylcholine receptor population in any region contained alpha5. Deletion of the alpha5 subunit gene resulted in localized loss of function (thalamus, striatum), which was itself confined to the DHbetaE-sensitive receptor population. No changes in receptor expression were seen. Consequently, functional changes must occur as a result of altered function per unit of receptor. The selective depletion of high agonist activation affinity sites results in overall nicotinic function being reduced, and increases the overall agonist activation affinity. Together, these results describe the receptor-level changes underlying altered behavioral responses to nicotine in nicotinic acetylcholine receptor alpha5 subunit-null mutants.

Interactions between alpha-conotoxin MI and the Torpedo marmorata receptor alpha-delta interface.

The muscle-type nicotinic receptor has two distinguishable acetylcholine binding sites at the alpha-gamma and alpha-delta subunit interfaces; alpha-conotoxins can bind them selectively. Moreover, we previously reported that alpha-conotoxin MI can interact with Torpedo californica and Torpedo marmorata receptors showing that conotoxins can also detect receptors from different species of the same genus [L. Cortez, S.G. del Canto, F. Testai, M.B. de Jiménez Bonino, Conotoxin MI inhibits the acetylcholine binding site of the Torpedo marmorata receptor, Biochem. Biophys. Res. Commun. 295 (2002) 791-795]. Herein, to identify T. marmorata receptor regions involved in alpha-conotoxin MI binding, a photoactivatable reagent was used and labeled sites were mapped by enzymatic proteolysis, MALDI-TOF-MS and Edman degradation. alpha-Conotoxin MI binding determinants were found and studies revealed a second binding motif at the alpha/delta interface. A proposal for receptor-toxin interaction is discussed based on experimental results and docking studies.