α3ß4 Acetylcholine Nicotinic Receptors Are Components of the Secretory Machinery Clusters in Chromaffin Cells.

The heteromeric assembly of α3 and ß4 subunits of acetylcholine nicotinic receptors (nAChRs) seems to mediate the secretory response in bovine chromaffin cells. However, there is no information about the localization of these nAChRs in relationship with the secretory active zones in this cellular model. The present work presents the first evidence that, in fact, a population of these receptors is associated through the F-actin cytoskeleton with exocytotic machinery components, as detected by SNAP-25 labeling. Furthermore, we also prove that, upon stimulation, the probability to find α3ß4 nAChRs very close to exocytotic events increases with randomized distributions, thus substantiating the clear dynamic behavior of these receptors during the secretory process. Modeling on secretory dynamics and secretory component distributions supports the idea that α3ß4 nAChR cluster mobility could help with improving the efficiency of the secretory response of chromaffin cells. Our study is limited by the use of conventional confocal microscopy; in this sense, a strengthening to our conclusions could come from the use of super-resolution microscopy techniques in the near future.

Natural Polyhydroxy Flavonoids, Curcuminoids, and Synthetic Curcumin Analogs as α7 nAChRs Positive Allosteric Modulators.

The α7 nicotinic acetylcholine receptor (α7 nAChR) is a ligand-gated ion channel that is involved in cognition disorders, schizophrenia, pain, and inflammation. Allosteric modulation of this receptor might be advantageous to reduce the toxicity in comparison with full agonists. Our previous results obtained with some hydroxy-chalcones, which were identified as positive allosteric modulators (PAMs) of α7 nAChR, prompted us to evaluate the potential of some structurally related naturally occurring flavonoids and curcuminoids and some synthetic curcumin analogues, with the aim of identifying new allosteric modulators of the α7 nAChR. Biological evaluation showed that phloretin, demethoxycurcumin, and bis-demethoxicurcuming behave as PAMs of α7 nAChR. In addition, some new curcumin derivatives were able to enhance the signal evoked by ACh; the activity values found for the tetrahydrocurcuminoid analog 23 were especially promising.

Acetylcholine nicotinic receptor subtypes in chromaffin cells.

In the adrenal gland, acetylcholine released on stimulation of the sympathetic splanchnic nerve activates neuronal-type nicotinic receptors (nAChRs) in chromaffin cells and triggers catecholamine secretion. At least two subtypes of nAChRs have been described in bovine chromaffin cells. The main subtype, a heteromeric assembly of α3, ß4 and perhaps α5 subunits, is involved in the activation step of the catecholamine secretion process and is not blocked by the snake toxin α-bungarotoxin. The other is α-bungarotoxin-sensitive, and its functional role has not yet been well defined. The α7 subunit conforms the homomeric structure of this subtype. All nAChR subunits share the same molecular organization and structural data at atomic resolution level are now available for some homomeric and heteromeric ensembles. The α3, ß4 and α5 subunits are clustered in genomes of different species, with the transcription factor Sp1 playing a co-ordinating role in the transcriptional regulation of these three subunits. The transcription factor Egr-1 controls the differential expression of α7 nAChR in adrenergic chromaffin cells, as happens with the enzyme phenylethanolamine N-methyl transferase. For unknown reasons, whole cell currents observed in bovine chromaffin cells clearly differ of the ones observed when different combinations of subunit RNAs are injected in oocytes. In addition to the typical nicotinic ligands, a variety of unrelated substances with clinical relevance can target nAChRs in chromaffin cells and, therefore, affect catecholamine secretion. They can act as agonists, antagonists or allosteric modulators.

Expression and functional properties of α7 acetylcholine nicotinic receptors are modified in the presence of other receptor subunits.

Although α7 nicotinic receptors are predominantly homopentamers, previous reports have indicated that α7 and ß2 subunits are able to form heteromers. We have studied whether other nicotinic receptor subunits can also assemble with α7 subunits and the effect of this potential association. Coexpression of α7 with α2, α3, or ß4 subunits reduced to about half, surface α-bungarotoxin binding sites and acetylcholine-gated currents. This is probably because of inhibition of membrane trafficking, as the total amount of α7 subunits was similar in all cases and a significant proportion of mature α7 receptors was present inside the cell. Only ß4 subunits appeared to directly associate with α7 receptors at the membrane and these heteromeric receptors showed some kinetic and pharmacological differences when compared with homomeric α7 receptors. Finally, we emulated the situation of bovine chromaffin cells in Xenopus laevis oocytes by using the same proportion of α3, ß4, α5, and α7 mRNAs, finding that α-bungarotoxin binding was similarly reduced in spite of increased currents, apparently mediated by α3ß4(α5) receptors.

Ric-3 promotes alpha7 nicotinic receptor assembly and trafficking through the ER subcompartment of dendrites.

The function of Ric-3, which is required for nicotinic acetylcholine receptor (nAChR) expression in C. elegans, is unclear. Here we found that Ric-3 can promote or inhibit cell-surface delivery of alpha-bungarotoxin-binding nAChRs (BgtRs) composed of alpha7 subunits. At low levels, Ric-3 promoted BgtR assembly, endoplasmic reticulum (ER) release, and cell-surface delivery without trafficking from the ER. At high Ric-3 levels, Ric-3 suppressed BgtR surface delivery, but not its assembly, and BgtRs were retained in the ER or in Ric-3-containing aggregates. In PC12 cells, native BgtRs trafficked to the cell surface from the ER where low levels of endogenous Ric-3 were observed. In cultured neurons, native Ric-3 levels were higher than in PC12 cells, and Ric-3 and alpha7 subunits were found in somata and dendrites, but not axons, of inhibitory interneurons. Ric-3 trafficked with alpha7 subunits in rapidly moving vesicles to dendrites, where it was restricted to the ER subcompartment. We conclude that Ric-3 has two potential functions. At low levels, Ric-3 interactions are short-lived and promote BgtR assembly and ER release. At higher levels, Ric-3 interactions are longer-lived and mediate ER retention. In neurons, Ric-3 ER retention appears to promote transport within the dendritic ER subcompartment, thereby restricting alpha7 trafficking to dendrites and preventing axonal transport.

Role of loop 9 on the function of neuronal nicotinic receptors.

We have studied the role of loop 9 in the function of neuronal nicotinic receptors. By systematically mutating the residues in the loop we have determined that the most important amino acids determining the coupling of binding to gating are the ones closer to the transmembrane region. Single mutations at location E173 in homomeric alpha7 receptors destroyed their function by completely abolishing the current while preserving the expression at the membrane. In contrast, heteromeric receptor alpha3beta4 with the same mutations retained some function. We conclude that loop 9 has a different role in the function of homomeric and heteromeric receptors.

Mutants of ß-strand ß3 and the loop B in the interface between α7 subunits of a homomeric acetylcholine receptor show functional and pharmacological alterations.

Activation of nicotinic acetylcholine receptors (nAChR) requires a global conformational change involving a number of domains of the protein. Structural data from Torpedo nAChR suggest that adjacent subunits might be functionally coupled at the interface between the ß-strand ß3 and the loop B through a salt bridge between α1Asp152 and γArg78. We have checked this hypothesis in homomeric α7 nAChRs by mutating residues at these (Gly152 and Arg79) and neighboring locations and analyzing the results obtained after expression of single and double mutants in Xenopus oocytes. We found that Arg79 mutants showed a decreased gating function when challenged with different agonists, being the reduction more important for dimethylphenylpiperazinium. EC(50) values in these mutants were also increased up to 30-fold. In contrast, mutating Gly152 only showed significant higher EC(50) values for ACh. However, all Gly153 mutants presented increased gating function and lower EC(50) values with no significant differences among them. When analyzing several mutant cycles it is concluded that Arg79 is functionally coupled to Gly152, but neither to Gly153 nor to Asp157. These data suggest an involvement of the minus side of homomeric α7 nAChRs in their gating function, reinforcing the significance of complementary subunits in the gating of neuronal nAChRs.

Substitutions of amino acids in the pore domain of homomeric α7 nicotinic receptors for analogous residues present in heteromeric receptors modify gating, rectification and binding properties.

We have studied the role of different amino acids in the M2 transmembrane domain of the α7 neuronal nicotinic receptor by mutating residues that differ from the ones located at the same positions in other α (α2-α10) or ß (ß2-ß4) subunits. Our aim was to investigate the contribution of these amino acids to the peculiar kinetic and inward rectification properties that differentiate the homomeric α7 receptor from other nicotinic receptors. Mutations of several residues strongly modified receptor function. We found that Thr245 had the most profound effect when mutated to serine, an amino acid present in all heteromeric receptors composed of α and ß subunits, by dramatically increasing the maximal current, decreasing the decaying rate of the currents and decreasing receptor rectification. Some mutants also showed altered agonist-binding properties as revealed by shifts in the dose-response curves for acetylcholine. We conclude that residues in the M2 segment and flanking regions contribute to the unusual properties of the α7 receptor, especially to its characteristic fast kinetic behavior and strong inward rectification and furthermore to the potency of agonists.

Binding-gating coupling in a nondesensitizing alpha7 nicotinic receptor A single channel pharmacological study.

The highly conserved alphaLys145 has been suggested to play an important role in the early steps of activation of the nicotinic acetylcholine receptor (nAChR) by acetylcholine. Both macroscopic and single channel currents were recorded in the slowly desensitizing mutants L248T- and K145A-L248T-alpha7 receptors expressed in Xenopus oocytes. On ACh-evoked currents, substitution of Lys145 by alanine showed the same effects that in wild type receptors: moderately decreased gating function and a more-than-expected loss of ACh potency, thus validating the experimental model. Single channel analysis quantitatively agreed with macroscopic data and revealed that impaired gating function in the double mutant alpha7K145A/L248T is the consequence of a slower opening rate, beta. Several nicotinic agonists were also studied, showing important features. Particularly, dimethylphenylpiperazinium (DMPP), acting as an antagonist in alpha7K145A, became a full agonist in alpha7K145A/L248T. Single channel analysis of DMPP-evoked currents showed effects of Lys145 removal similar to those observed with ACh. Data suggest that alpha7Lys145 facilitates the early steps of channel activation. Moreover, the slowly desensitizing mutant alpha7L248T could be an interesting tool for the study of channel activation in alpha7 receptors. Nevertheless, its extensively altered pharmacology precludes the simple extrapolation of pharmacological data obtained in singly mutated alpha7 receptors.

The loop between beta-strands beta 2 and beta 3 and its interaction with the N-terminal alpha-helix is essential for biogenesis of alpha 7 nicotinic receptors.

Recently, we have shown that the alpha-helix present at the N-termini of alpha7 nicotinic acetylcholine receptors plays a crucial role in their biogenesis. Structural data suggest that this helix interacts with the loop linking beta-strands beta2 and beta3 (loop 3). We studied the role of this loop as well as its interaction with the helix in membrane receptor expression. Residues from Asp62 to Val75 in loop 3 were mutated. Mutations of conserved amino acids, such as Asp62, Leu65 and Trp67 abolished membrane receptor expression in Xenopus oocytes. Others mutations, at residues Asn68, Ala69, Ser70, Tyr72, Gly74, and Val 75 were less harmful although still produced significant expression decreases. Steady state levels of wild-type and mutant alpha7 receptors (L65A, W67A, and Y72A) were similar but the formation of pentameric receptors was impaired in the latter (W67A). Mutation of critical residues in subunits of heteromeric nicotinic acetylcholine receptors (alpha3beta4) also abolished their membrane expression. Complementarity between the helix and loop 3 was evidenced by studying the expression of chimeric alpha7 receptors in which these domains were substituted by homologous sequences from other subunits. We conclude that loop 3 and its docking to the alpha-helix is an important requirement for receptor assembly.

Role of the extracellular transmembrane domain interface in gating and pharmacology of a heteromeric neuronal nicotinic receptor.

Nicotinic acetylcholine receptors (nAChRs) transmit the agonist signal to the channel gate through a number of extracellular domains. We have previously shown that particular details of the process of coupling binding to gating could be quantitative and qualitatively different in muscle and neuronal type nAChRs. We have extended previous studies on homomeric alpha7 nAChRs to heteromeric alpha3beta4 nAChRs, by mutating residues located at loops 2 and 7, and M2-M3 linker of both alpha3 and beta4 subunits which, in order to monitor surface expression, were modified to bind alpha-bungarotoxin, and expressed in Xenopus oocytes. We show that, in general, mutations in these domains of both alpha3 and beta4 subunits affect the gating function, although the effects are slightly larger if they are inserted in the alpha3 subunit. However, the involvement of a previously reported intrasubunit interaction in coupling (Gln48-Ile130) seems to be restricted to the beta4 subunit. We also show that mutations at these domains, particularly loop 2 of the alpha3 subunit, change the pharmacological profile of alpha3beta4 nAChRs, decreasing nicotine's and increasing cytisine's effectiveness relative to acetylcholine. It is concluded that, unlike muscle nAChRs, the non-alpha subunits play a relevant role in the coupling process of neuronal alpha3beta4 nAChRs.

Role of the N-terminal alpha-helix in biogenesis of alpha7 nicotinic receptors.

We studied the role of the alpha-helix present at the N-terminus of nicotinic acetylcholine receptor (nAChR) subunits in the expression of functional channels. Deletion of this motif in alpha7 subunits abolished expression of nAChRs at the membrane of Xenopus oocytes. The same effect was observed upon substitution by homologous motifs of other ligand-gated receptors. When residues from Gln4 to Tyr15 were individually mutated to proline, receptor expression strongly decreased or was totally abolished. Equivalent substitutions to alanine were less harmful, suggesting that proline-induced break of the alpha-helix is responsible for the low expression. Steady-state levels of wild-type and mutant subunits were similar but the formation of pentameric receptors was impaired in the latter. In addition, those mutants that reached the membrane showed a slightly increased internalization rate. Expression of alpha7 nAChRs in neuroblastoma cells confirmed that mutant subunits, although stable, were unable to reach the cell membrane. Analogous mutations in heteromeric nAChRs (alpha3beta4 and alpha4beta2) and 5-HT(3A) receptors also abolished their expression at the membrane. We conclude that the N-terminal alpha-helix of nAChRs is an important requirement for receptor assembly and, therefore, for membrane expression.

1-(2',5'-Dihydroxyphenyl)-3-(2-fluoro-4-hydroxyphenyl)-1-propanone (RGM079): A Positive Allosteric Modulator of α7 Nicotinic Receptors with Analgesic and Neuroprotective Activity.

Acetylcholine α7 nicotinic receptors are widely expressed in the brain, where they are involved in the central processing of pain as well as in neuropsychiatric, neurodegenerative, and inflammatory processes. Positive allosteric modulators (PAMs) show the advantage of allowing the selective regulation of different subtypes of acetylcholine receptors without directly interacting with the agonist binding site. Here, we report the preparation and biological activity of a fluoro-containing compound, 1-(2',5'-dihydroxyphenyl)-3-(2-fluoro-4-hydroxyphenyl)-1-propanone (8, RGM079), that behaves as a potent PAM of the α7 receptors and has a balanced pharmacokinetic profile and antioxidant properties comparable or even higher than well-known natural polyphenols. In addition, compound RGM079 shows neuroprotective properties in Alzheimer's disease (AD)-toxicity related models. Thus, it causes a concentration-dependent neuroprotective effect against the toxicity induced by okadaic acid (OA) in the human neuroblastoma cell line SH-SY5Y. Similarly, in primary cultures of rat cortical neurons, RGM079 is able to restore the cellular viability after exposure to OA and amyloid peptide Aß1-42, with cell death almost completely prevented at 10 and 30 µM, respectively. Finally, compound RGM079 shows in vivo analgesic activity in the complete Freund's adjuvant (CFA)-induced paw inflammation model after intraperitoneal administration.

Interactions between loop 5 and beta-strand beta6' are involved in alpha7 nicotinic acetylcholine receptors channel gating.

Binding of agonists to nicotinic acetylcholine receptors (nAChR) is coupled to channel opening through local rearrangements of different domains of the protein. Recent structural data suggest that two of these regions could be the loop 5 (L5) and the beta-strand beta6', both forming the inner part of the N-terminal domain. Amino acids in these domains were mutated in alpha7 nAChRs, and expression levels and functional responses of mutant receptors were measured. Mutations located at the putative apex of L5, Asp97 and Glu98, and also at Phe100, gave receptors with smaller currents, showing qualitative differences with respect to muscle nAChRs. In contrast, mutations in the beta-strand beta6' (at Phe124 and Lys125) showed increased functional responses. Mutations affected equally the responses to acetylcholine and dimethylphenylpiperazinium, except in Phe100 where the latter was sevenfold less effective than in wild-type. Currents in mutants decayed with almost the same kinetics, ruling out large effects on desensitization. Analysis of double mutants demonstrated a functional coupling among the three electrically charged amino acids Asp97, Glu98, and Lys125, and also between Phe100 and Phe124. The results are compatible with the involvement of functional interactions between L5 and beta-strand beta6' during nAChR activation.

Molecular characterization and localization of the RIC-3 protein, an effector of nicotinic acetylcholine receptor expression.

The RIC-3 protein acts as a regulator of acetylcholine nicotinic receptor (nAChR) expression. In Xenopus laevis oocytes the human RIC-3 (hRIC-3) protein enhances expression of alpha7 receptors and abolishes expression of alpha4beta2 receptors. In vitro translation of hRIC-3 evidenced its membrane insertion but not the role as signal peptide of its first transmembrane domain (TMD). When the TMDs of hRIC-3 were substituted, its effects on nAChR expression were attenuated. A certain linker length between the TMDs was also needed for alpha7 expression enhancement but not for alpha4beta2 inhibition. A combination of increased alpha7 receptor steady state levels, facilitated transport and reduced receptor internalization appears to be responsible for the increase in alpha7 membrane expression induced by hRIC-3. Antibodies against hRIC-3 showed its expression in SH-SY5Y and PC12 cells and its induction upon differentiation. Immunohistochemistry demonstrated the presence of RIC-3 in rat brain localized, in general, in places where alpha7 nAChRs were found.

Functional evidence for the inflammatory reflex in teleosts: A novel α7 nicotinic acetylcholine receptor modulates the macrophage response to dsRNA.

The inflammatory reflex modulates the innate immune system, keeping in check the detrimental consequences of overstimulation. A key player controlling the inflammatory reflex is the alpha 7 acetylcholine receptor (α7nAChR). This receptor is one of the signalling molecules regulating cytokine expression in macrophages. In this study, we characterize a novel teleost α7nAChR. Protein sequence analysis shows a high degree of conservation with mammalian orthologs and trout α7nAChR has all the features and essential amino acids to form a fully functional receptor. We demonstrate that trout macrophages can bind α-bungarotoxin (α-BTX), a competitive antagonist for α7nAChRs. Moreover, nicotine stimulation produces a decrease in pro-inflammatory cytokine expression after stimulation with poly(I:C). These results suggest the presence of a functional α7nAChR in the macrophage plasma membrane. Further, in vivo injection of poly(I:C) induced an increase in serum ACh levels in rainbow trout. Our results manifest for the first time the functional conservation of the inflammatory reflex in teleosts.

Amino acid and peptide prodrugs of diphenylpropanones positive allosteric modulators of α7 nicotinic receptors with analgesic activity.

α7 Nicotinic acetylcholine receptors (nAChRs) are ion channels implicated in a number of CNS pathological processes, including pain and psychiatric, cognitive and inflammatory diseases. Comparing with orthosteric agonism, positive allosteric modulation of these channels constitutes an interesting approach to achieve selectivity versus other nicotinic receptors. We have recently described new chalcones and 1,3-diphenylpropanones as positive allosteric modulators (PAMs) of α7 nAChRs, which proved to have good analgesic activities but poor pharmacokinetic properties. Here we report the preparation of amino acid and peptide derivatives as prodrugs of these modulators with the aim of improving their in vivo biological activity. While the valine derivative showed very short half life in aqueous solutions to be considered a prodrug, Val-Val and Val-Pro-Val are suitable precursors of the parent 1,3-diphenylpropanones, via chemical and enzymatic transformation, respectively. Compounds 19 (Val-Val) and 21 (Val-Pro-Val), prodrugs of the 2',5',4-trihydroxy-1,3-diphenylpropan-1-one 3, showed significant antinociceptive activity in in vivo assays. The best compound, 21, displayed a better profile in the analgesia test than its parent compound 3, exhibiting about the same potency but long-lasting effects.

Improved gating of a chimeric alpha7-5HT3A receptor upon mutations at the M2-M3 extracellular loop.

Acetylcholine-evoked currents of the receptor chimera alpha7-5HT3A V201 expressed in Xenopus oocytes are strikingly small when compared to the amount of alpha-bungarotoxin binding sites detected at the oocyte membrane. Since the chimeric receptor is made of the extracellular N-terminal region of the rat alpha7 nicotinic acetylcholine receptor and the C-terminal region of the mouse 5-HT3A receptor, which includes the ion channel, we hypothesized that communication between these two regions was not optimal. Here, we show that mutating to aspartate several adjacent positions in the M2-M3 extracellular linker increases current amplitudes to different extents, thus confirming the important role of this region on receptor gating.

Role of the RIC-3 protein in trafficking of serotonin and nicotinic acetylcholine receptors.

Neurotransmitter-gated receptors are assembled in the endoplasmic reticulum and transported to the cell surface through a process that might be of central importance to regulate the efficacy of synaptic transmission (Kneussel and Betz, 2000; Kittler and Moss, 2003). This process is relatively inefficient- what may be the consequence of tight quality controls that guarantee the functional competence of the final product. For this purpose, specific proteins involved in assembly and trafficking of receptors might be required (Keller and Taylor, 1999; Millar, 2003; Wanamaker et al., 2003). The RIC-3 protein could be one of them, as mutations in the ric-3 gene affect maturation of nicotinic acetylcholine receptors (nAChRs) in Caenorhabditis elegans (Halevi et al., 2002). Moreover, the human homolog hRIC-3 showed differential effects when coexpressed with several ligand-gated receptors (Halevi et al., 2003). Thus, it enhanced alpha7 nAChR expression while inhibiting expression of other nAChR subtypes (alpha4beta2 and alpha3beta4) and 5-HT3 serotonin receptors (5-HT3Rs). These opposite effects suggested that the RIC-3 protein might play a key role in the biogenesis of some ligand-gated receptors and prompted us to investigate how it performs its action. Here, we show that the RIC-3 protein acts as a barrier for some receptors like alpha4beta2 nAChRs and 5-HT3Rs, stopping the traffic of mature receptors to the membrane. In contrast, the inefficient transport of alpha7 nAChRs is enhanced by RIC-3 in a process in which certain amino acids at the amphipathic helix located at the C-terminal region of the large cytoplasmic domain are involved.

Activation and blockade by choline of bovine alpha7 and alpha3beta4 nicotinic receptors expressed in oocytes.

Choline, the precursor and the metabolite of acetylcholine, is reputed as a selective alpha7 nicotinic receptor agonist. In this study, however, we have seen that choline exerted a dual effect on bovine nicotinic receptors expressed in Xenopus oocytes. On the one hand, choline behaved as a weak full agonist on bovine alpha7-mediated inward currents, with an EC50 of 0.43 mM. On the other, choline blocked bovine alpha3beta4 currents, with an IC50 of 0.97 mM. The blockade by choline was fast (tau(on), 0.36 s), fully reversible (tau(off), 1.23 s), exhibited voltage-dependence (60% blockade at -100 mV and 30% blockade at -40 mV), and was of a non-competitive nature, suggesting an open-channel type of alpha3beta4 receptor blockade. Thus, choline by activating alpha7 receptors and/or blocking alpha3beta4 receptors might play a physiological role in the control of neurotransmission at cholinergic synapses where alpha7 and alpha3beta4 receptor are expressed.