Salvador Sala

• Miguel Hernández University of Elche

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 i...

Moral judgments are typically explained by a combination of either deontological considerations about the nature of actions, or quantitative assessments of the consequences of those actions. These proposals, however, have serious limitations such as being insensitive to personal biases and global circumstances. This study presents an alternative ap...

The regenerative activity of adult stem cells carries a risk of cancer, particularly in highly renewable tissues. Members of the family of inhibitor of apoptosis proteins (IAPs) inhibit caspases and cell death, and are often deregulated in adult cancers; however, their roles in normal adult tissue homeostasis are unclear. Here, we show that regulat...

The regenerative activity of adult stem cells carries a high risk of cancer, particularly in highly renewable tissues. To guarantee that the correct organ size is attained and to cope with the continual risk of cancer, developing tissues often use programmed cell death (PCD) as an adaptive mechanism to cull excess and abnormal cells. Members of the...

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 nicotin...

Noise and artifacts are inherent contaminating components and are particularly present in Holter electrocardiogram (ECG) monitoring. The presence of noise is even more significant in long-term monitoring (LTM) recordings, as these are collected for several days in patients following their daily activities; hence, strong artifact components can temp...

The problem of sending information at long distances, without significant attenuation and at a low cost, is common to both artificial and natural environments. In the brain, a widespread strategy to solve the cost-efficiency trade off in long distance communication is the presence of convergent pathways, or bottlenecks. In the visual system, for ex...

Objective noise metrics such as the signal to noise ratio and the root mean squared value, are not always related to the actual impact that noise can have on the clinical evaluation of cardiac signals. This work is intended to design a database along with a set of criteria to be used as an initial solid gold-standard of noise severity within a scal...

A series of multi-target directed propargylamines, as well as other differently susbstituted piperidines have been screened as potential modulators of neuronal nicotinic acetylcholine receptors (nAChRs). Most of them showed antagonist actions on α7 nAChRs. Especially, compounds 13, 26 and 38 displayed sub-micromolar IC50 values on homomeric α7 nACh...

Results: According to their substituents, 1,3-diphenylpropan-1-one derivatives act as α7 nAChRs negative allosteric modulators (NAM, OMe) or Type I positive allosteric modulators (PAMs, OH). Compounds 7 and 31 were the most effective (989 and 666% enhancement of ACh-induced currents) and potent (EC50: 12.9 and 6.85 μM) PAMs. They exhibited strong...

We have characterized the effect of triazine derivatives on neuronal nicotinic receptors expressed in Xenopus oocytes. All triazines investigated inhibit the current of α7 and α3β4 neuronal nicotinic receptors elicited by acetylcholine. The effect is concentration dependent, reversible, and noncompetitive. In contrast, some derivatives have a dual...

Although α7 nicotinic receptors are predominantly homopentamers, previous reports have indicated that α7 and β2 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 β4 subunits reduced to abo...

Deletion of a small cytoplasmic fragment close to the fourth transmembrane segment of the nicotinic α7 receptor (Glu437 to Arg447) abolished membrane expression. Different single mutants showed moderate to strong decreases in expression whereas the latter was totally abolished upon proline substitutions. We hypothesize that preservation of an α-hel...

J. Neurochem. (2011) 119, 40–49. 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 β (β2–β4) subunits. Our aim was to investigate the contribution of these amino acids to the peculi...

J. Neurochem. (2011) 118, 968–978. 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 β-strand β3 and the loop B through a salt bridge...

J. Neurochem. (2010) 113, 1036–1045. 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....

J. Neurochem. (2010) 113, 1036–1045. 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 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...

J. Neurochem. (2010) 112, 103–111. Recently, we have shown that the α-helix present at the N-termini of α7 nicotinic acetylcholine receptors plays a crucial role in their biogenesis. Structural data suggest that this helix interacts with the loop linking β-strands β2 and β3 (loop 3). We studied the role of this loop as well as its interaction with...

We have studied the role of the highly conserved residue alphaLysine145 in the early steps of activation by acetylcholine of the nicotinic acetylcholine receptor (nAChR). Both macroscopic and single-channel currents were recorded in the slowly desensitizing chimeric mutant receptor alpha7V201-5HT3A/R432Q/R436D/R440A, made of alpha7 nAChRs and serot...

To test the hypothesis that cell-dependent expression of alpha7 receptors is due to differences in protein folding or assembly, we constructed a chimeric rat alpha7 subunit with green fluorescent protein (GFP) at the receptor C-terminal. Expression of alpha7-GFP in Xenopus oocytes resulted in currents that were indistinguishable from wild type rece...

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 liga...

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-e...

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...

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 doma...

Binding of agonists to nicotinic acetylcholine receptors results in channel opening. Previously, we have shown that several charged residues at three different domains of the alpha7 nicotinic receptor are involved in coupling binding and gating, probably through a network of electrostatic interactions. This network, however, could also be integrate...

We studied the role of the cytoplasmic regions adjacent to the M3 and M4 transmembrane segments of alpha7 nicotinic receptors in the expression of functional channels. For this purpose, a total of 50 amino acids were mutated throughout the mentioned regions. Mutants close to M3, from Arg294 to Leu321, showed slight modifications in the levels of al...

Chromaffin cells from the bovine adrenal medulla express α-bungarotoxin-sensitive acetylcholine receptors whose subunit composition is unknown. Northern blot analysis showed that the α7 subunit, a main component of these α-bungarotoxin-sensitive acetylcholine receptors in avian and rat brain, is expressed in chromaffin cells. The cDNA of this bovin...

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 qualit...

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 t...

Using a yeast two-hybrid screening we report the isolation of a novel human protein, hCRELD2beta, that interacts specifically with the large cytoplasmic regions of human nicotinic acetylcholine receptor (nAChR) alpha4 and beta2 subunits, both in yeast cells and in vitro. This interaction is not detected with nAChR alpha7 and alpha3 subunits. The hC...

Activation of nicotinic acetylcholine receptors is initiated by binding of agonists, and as a consequence, specific domains transmit the chemical signal to the channel gate through a sequence of conformational changes. Recent high-resolution structural data from a snail acetylcholine binding protein have shown that the side chain of a lysine residu...

The ric-3 gene is required for maturation of nicotinic acetylcholine receptors in Caenorhabditis elegans. The human homolog of RIC-3, hRIC-3, enhances expression of alpha7 nicotinic receptors in Xenopus laevis oocytes, whereas it totally abolishes expression of alpha4beta2 nicotinic and 5-HT3 serotonergic receptors. Both the N-terminal region of hR...

Binding of agonists to nicotinic acetylcholine receptors generates a sequence of conformational changes resulting in channel opening. Previously, we have shown that the aspartate residue Asp-266 at the M2-M3 linker of the alpha7 nicotinic receptor is involved in connecting binding and gating. High resolution structural data suggest that this region...

The effects of various Flustra foliacea metabolites on different types of human neuronal nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes were investigated. Whereas most of the compounds tested had a small blocking effect, one of them, deformylflustrabromine, selectively increased the current obtained in alpha4beta2 receptors...

In Caenorhabditis elegans, the ric-3 gene is required for the maturation of multiple nicotinic acetylcholine receptors (nAChRs), whereas other neurotransmittergated channels expressed within the same cells are unaffected by the presence of RIC-3. Here we show that RIC-3 is a member of a conserved gene family with representatives in both vertebrates...

Sensory endings of nerve fibers signalling joint pain express stretch-activated ion channels which open in response to an increase in membrane tension. These stretch-activated channels have been well characterised in Xenopus laevis oocytes. We have investigated whether hylan solutions of different elastoviscosity used for intra-articular treatment...

Activation by noxious mechanical stimuli of sensory nerve fibres that signal joint pain takes place through stretch-activated ion channels, which open in response to increased membrane tension. It has been suggested that the analgesic effect of hyaluronan solutions used for intra-articular treatment of joint pain in humans are mediated by a reducti...

The role of the large intracellular loop of the nicotinic acetylcholine receptor (nAChR) alpha7 subunit in the expression of functional channels was studied. For this purpose, systematic deletions and substitutions were made throughout the loop and the ability of the mutated alpha7 subunits to support expression of functional nAChRs at the Xenopus...

Ginseng saponins, major active components of ginseng root used by folk medicine in the treatment of various diseases, produce multiple pharmacological responses having many effects on the central and peripheral nervous system. Specifically, ginsenoside Rg(2) has been shown to block the nicotinic acetylcholine receptors in bovine chromaffin cells. W...

We have investigated the effect of diltiazem and its newly synthesized derivative (+,−)-trans-3-acetoxy-8-chloro-2,3-dihydro-5[2-diisopropylamine)ethyl]-2-(4-methoxyphenyl)-1,5-benzothiazepin-4-(5H)-ona hydrochloride (JAC-65) on several recombinant human neuronal nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes. At 3 μM, both...

We investigated the effects of ginsenosides, the active ingredient of ginseng, on neuronal or muscle-type nicotinic acetylcholine receptor channel activity expressed in Xenopus oocytes after injection of cRNA encoding bovine neuronal alpha3beta4, alpha7 or human muscle alphabetadeltavarepsilon subunits. Treatment with acetylcholine elicited an inwa...

We have examined the role of a highly conserved arginine (R209), which flanks the M1 transmembrane segment of nAChRs, in the biogenesis and function of neuronal nAChRs. Point mutations revealed that, in alphaBgtx-sensitive neuronal alpha7 nAChRs, the conserved arginine is required for the transport of assembled receptors to the cell surface. By con...

Previous studies have shown that the gating mechanism of α3β4 neuronal nicotinic receptors is affected by a residue in the middle of the M2-M3 loop of the β4 subunit. We have extended the study of the same location to the α3 subunit. Bovine α3β4 receptors were mutated in position 268, substituting the residue present in wild-type receptors, i.e. le...

Previous studies have shown that the gating mechanism of alpha3beta4 neuronal nicotinic receptors is affected by a residue in the middle of the M2-M3 loop of the beta4 subunit. We have extended the study of the same location to the alpha3 subunit. Bovine alpha3beta4 receptors were mutated in position 268, substituting the residue present in wild-ty...

Previous studies have shown that the gating mechanism of α3β4 neuronal nicotinic receptors is affected by a residue in the middle of the M2-M3 loop of the β4 subunit. We have extended the study of the same location to the α3 subunit. Bovine α3β4 receptors were mutated in position 268, substituting the residue present in wild-type receptors, i.e. le...

Previous studies have shown that the gating mechanism of &#333Ţ neuronal nicotinic receptors is affected by a residue in the middle of the M2-M3 loop of the Ţ subunit. We have extended the study of the same location to the ō subunit. Bovine &#333Ţ receptors were mutated in position 268, substituting the residue present in wild-type receptors, i.e....

An aspartate residue in the M2-M3 loop of neuronal nicotinic receptor alpha7 subunits is a major determinant of the channel functional response. This residue is conserved in most beta4 subunits, e.g. human and rat, but not in others, e.g. bovine. We have used these differences to examine the mechanism by which this residue alters the functional pro...

The involvement of some structural domains in the gating of the neuronal nicotinic acetylcholine receptor (AChR) was studied by expressing functional alpha7/alpha3 chimeric subunits in Xenopus oocytes. Substitution of the M3 transmembrane segment in the alpha7 subunit modifies the kinetic properties of the chimeric AChRs as follows: (a) a 6-fold re...

Neuronal nicotinic acetylcholine receptors from bovine adrenomedullary chromaffin cells play a primary role in triggering catecholamine secretion. In the present study, their constituent subunits were characterized. In addition to the alpha 3 subunit, which we have previously cloned, the presence of alpha 5 and beta 4 but not of beta 2 subunits was...

The neuronal nicotinic acetylcholine receptor (nAChR) subunits alpha3 and alpha7 have different assembly behavior when expressed in heterologous expression systems: alpha3 subunits require other subunits to assemble functional nAChRs, whereas alpha7 subunits can produce homomeric nAChRs. A previous analysis of alpha7/alpha3 chimeric constructs iden...

Binding of agonists to nicotinic acetylcholine receptors generates a sequence of changes that activate a cation-selective conductance. By measuring electrophysiological responses in chimeric alpha7/alpha3 receptors expressed in Xenopus oocytes, we have showed the involvement of the M2-M3 loop in coupling agonist binding to the channel gate. An aspa...

Chromaffin cells from the bovine adrenal medulla express alpha-bungarotoxin-sensitive acetylcholine receptors whose subunit composition is unknown. Northern blot analysis showed that the alpha 7 subunit, a main component of these alpha-bungarotoxin-sensitive acetylcholine receptors in avian and rat brain, is expressed in chromaffin cells. The cDNA...

Differential expression of subunit genes from the nicotinic acetylcholine receptor (AChR) superfamily yields distinct receptor subtypes. As each AChR subtype has a specific subunit composition and many subunit combinations appear not to be expressed, each subunit must contain some information leading to proper assembly. The neuronal AChR subunits a...

Using a cDNA library from bovine adrenal medulla, and, subsequently, a bovine genomic library, we have isolated the gene coding for a non inactivating potassium channel. This gene encodes a 597-amino acid protein which we have called BAK5 as its sequence is very similar to members of Kv1.5 potassium channel family. Neuroblastoma cells (Neuro-2a cel...

Differential expression of subunit genes from the nicotinic acetylcholine receptor (AChR) superfamily yields distinct receptor subtypes. As each AChR subtype has a specific subunit composition and many subunit combinations appear not to be expressed, each subunit must contain some information leading to proper assembly. The neuronal AChR subunits a...

The use of voltage clamp with a single electrode has been useful in estimating kinetic parameters for a number of ionic whole-cell currents. There are two main types of such a technique: discontinuous voltage clamp (dSEVC) (Brennecke and Lindemann, 1974), and continuous voltage clamp (cSEVC) (Hamill et al., 1981). We have studied, by means of compu...

Potassium currents of cultured bovine chromaffin cells in the presence of CdCl2 (0.5 mM) or CoCl2 (2 - 4 mM) may be described as a component that follows an n4 kinetics over the first 100 ms. However, during prolonged voltage clamp steps (5 - 20 s) this current reaches a peak and then declines to a non-zero steady-state level. In the absence of pot...

There is ample physiological evidence for the presence of muscarinic acetylcholine receptors in pancreatic β-cells7,8,9,12. Stimulation of parasympathetic nerves can cause hypoglycemia and insulin release, an effect which may participate in the so-called cephalic phase of insulin secretion. Since the role of parasympathetic innervation in health an...