Francisco Sala

Universidad Miguel Hernández de Elche, Elx, Valencia, Spain

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Publications (59)238.76 Total impact

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    ABSTRACT: 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 effect on α4β2 receptors, by potentiating the currents at intermediate concentration and causing inhibition at higher concentrations. Triazine derivatives also affect the macroscopic kinetics of the heteromeric receptors α3β4 and α4β2 accelerating the rise and decay time course of the currents, but have no significant effect on the kinetics of homomeric α7 receptors. Two simple kinetic models are presented. The first reproduces the effects of different concentrations of triazines both on the peak currents and on the macroscopic kinetics of α7 with a simple inhibitory result. The second model describes the behavior of α4β2 receptors involving a more complex dual action.
    ACS Chemical Neuroscience 07/2014; · 3.87 Impact Factor
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    ABSTRACT: 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 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 β4 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, β4, α5, and α7 mRNAs, finding that α-bungarotoxin binding was similarly reduced in spite of increased currents, apparently mediated by α3β4(α5) receptors.
    Journal of Neurochemistry 08/2012; 123(4):504-14. · 3.97 Impact Factor
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    ABSTRACT: 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-helix formed by the fourth transmembrane segment and the adjacent cytoplasmic region is essential for membrane receptor expression. Moreover, in selected mutants with low or null membrane expression, a significant proportion of mature receptors was present inside the cell. Hence, elements in this cytoplasmic fragment might influence receptor transport to the membrane.
    FEBS letters 08/2011; 585(15):2477-80. · 3.54 Impact Factor
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    ABSTRACT: 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 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 β 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.
    Journal of Neurochemistry 07/2011; 119(1):40-9. · 3.97 Impact Factor
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    ABSTRACT: 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 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.
    Journal of Neurochemistry 07/2011; 118(6):968-78. · 3.97 Impact Factor
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    ABSTRACT: 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.
    Journal of Neurochemistry 05/2010; 113(4):1036-45. · 3.97 Impact Factor
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    ABSTRACT: 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.
    Biochimica et Biophysica Acta 03/2010; 1798(3):654-9. · 4.66 Impact Factor
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    ABSTRACT: 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.
    Journal of Neurochemistry 10/2009; 112(1):103-11. · 3.97 Impact Factor
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    ABSTRACT: 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 serotonin receptors of subtype 3A (ch1), and its corresponding mutant K145A (ch1/K145A) expressed in Xenopus oocytes. Mutant ch1/K145A receptors had a reduced gating function similar to that produced by the same mutation in the wild type receptor alpha7. The mutated receptor has reduced opening rate constants, beta, and increased closing rate constants, alpha.
    FEBS letters 03/2009; 583(6):1045-51. · 3.54 Impact Factor
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    ABSTRACT: 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 receptors but were only 33% of control. (125)I-alpha-bungarotoxin (alphaBGT) binding at the oocyte surface was reduced to 23% of wild type. Transfection of alpha7-GFP into GH4C1 cells produced fluorescence that was less intense than GFP alone, but showed significant alpha-BGT binding compared to transfection with GFP. In contrast, alpha7-GFP transfection in SH-EP1, HEK293 and CHO-CAR cells produced fluorescence without alphaBGT binding. Flow cytometry of cells transfected with alpha7-GFP indicated fluorescence in both SH-EP1 and GH4C1 cells, but surface toxin binding sites and sites immunoprecipitated using anti-GFP antibodies were undetectable in SH-EP1 cells, suggesting a problem in folding/assembly rather than trafficking. Surprisingly, integrated fluorescence intensities in GH4C1 cells transfected with alpha7-GFP did not correlate with amounts of cell surface or immunoprecipitable alphaBGT binding. Therefore, GFP folding at the C-terminal of the alpha7-GFP chimera is cell-line independent, but toxin binding is highly cell-line dependent, suggesting that if altered protein folding is involved in the cell-type dependence of alpha7 receptor expression, the phenomenon is restricted to specific protein domains. Further, C-terminal GFP-labeled alpha7 receptors decreased the efficiency of folding/assembly not only of chimeric subunits, but also wild-type subunits, suggesting that the C-terminal is an important domain for alpha7 receptor assembly.
    Brain research 03/2009; 1259:7-16. · 2.46 Impact Factor
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    ABSTRACT: 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.
    Journal of Neurochemistry 02/2009; 108(6):1399-409. · 3.97 Impact Factor
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    ABSTRACT: 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.
    Biochimica et Biophysica Acta 12/2008; 1788(2):410-6. · 4.66 Impact Factor
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    ABSTRACT: 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.
    Journal of Neurochemistry 06/2008; 105(3):617-27. · 3.97 Impact Factor
  • F Sala, A Nistri, M Criado
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    ABSTRACT: In the adrenal medulla, acetylcholine released by the sympathetic splanchnic nerves activates neuronal-type nicotinic acetylcholine receptors (nAChRs) on the membrane of chromaffin cells which liberate catecholamines into the bloodstream in preparation for the fight and flight reactions. On adrenal chromaffin cells the main class of nAChRs is a pentameric assembly of alpha3 and beta4 subunits that forms ion channels which produce membrane depolarization by increasing Na+, K+ and Ca2+ permeability. Homomeric alpha7 nicotinic receptors are expressed in a species-dependent manner and do not contribute to catecholamine secretion. Chromaffin cell nAChRs rapidly activate and desensitize with full recovery on washout. nAChR activity is subjected to various types of dynamic regulation. It is allosterically modulated by the endogenous neuropeptide substance P that stabilizes receptors in their desensitized state, thus depressing their responsiveness. The full-length peptide CGRP acts as a negative allosteric modulator by inhibiting responses without changing desensitization, whereas its N-terminal fragments act as positive allosteric modulators to transiently enhance nAChR function. nAChR expression increases when cells are chronically exposed to either selective antagonists or agonists such as nicotine, a protocol mimicking the condition of chronic heavy smokers. In this case, large upregulation of nAChRs occurs even though most of the extra nAChRs remain inside the cells, creating a mismatch between the increase in total nAChRs and increase in functional nAChRs on the cell surface. These findings highlight the plastic properties of cholinergic neurotransmission in the adrenal medulla to provide robust mechanisms for adapting catecholamine release to acute and chronic changes in sympathetic activity.
    Acta Physiologica 03/2008; 192(2):203-12. · 4.38 Impact Factor
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    ABSTRACT: 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.
    Journal of Neurochemistry 03/2008; 104(3):719-30. · 3.97 Impact Factor
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    ABSTRACT: 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 integrated by other residues. To test this hypothesis, non-charged amino acids were mutated and expression levels and electrophysiological responses of mutant receptors were determined. Mutants at positions Asn47 and Gln48 (loop 2), Ile130, Trp134, and Gln140 (loop 7), and Thr264 (M2-M3 linker) showed poor or null functional responses, despite significant membrane expression. By contrast, mutants F137A and S265A exhibited a gain of function effect. In all cases, changes in dose-response relationships were small, EC(50) values being between threefold smaller and fivefold larger, arguing against large modifications of agonist binding. Peak currents decayed at the same rate in all receptors except two, excluding large effects on desensitization. Thus, the observed changes could be mostly caused by alterations of the gating characteristics. Moreover, analysis of double mutants showed an interconnection between some residues in these domains, especially Gln48 with Ile130, suggesting a potential coupling between agonist binding and channel gating through these amino acids.
    Journal of Neurochemistry 11/2007; 103(2):725-35. · 3.97 Impact Factor
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    ABSTRACT: 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 alpha-bungarotoxin binding sites and acetylcholine-evoked currents. Exceptions were mutants located at two clusters (His296 to Pro300 and Ile312 to Trp316), which exhibited low expression levels. In addition, some mutants showed altered functional responses. Many mutants close to M4 showed increased receptor expression, especially the ones located at the hydrophobic face of a putative amphipathic helix. This effect seems to be the consequence of a combination of increased receptor biosynthesis, higher transport efficiency and delayed degradation, such that we postulate that elements in the amphipathic domain strongly influence receptor stability. Finally, some mutants in this region showed altered functional responses: elimination of positively charged residues (Arg424 and Arg426) increased currents, whereas the opposite was observed upon suppression of negatively charged ones (Glu430 and Glu432). These results suggest that the cytoplasmic regions close to M3 and M4 play important structural and functional roles.
    Journal of Neurochemistry 02/2007; 100(2):406-15. · 3.97 Impact Factor
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    ABSTRACT: 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 bovine α7 subunit was cloned by polymerase chain reaction amplification of adrenal medulla RNA for detailed characterization of structure and function. The protein-coding region revealed 92% amino acid sequence identity to rat α7 and 89% to chicken α7 subunits. The α-bungarotoxin affinity of α7 homomers expressed in Xenopus oocytes was similar to that observed previously with native chromaffin α-bungarotoxin sensitive acetylcholine receptors. Cross-linking and sucrose gradient experiments suggested that, like the muscular and neuronal acetylcholine receptors, the α7 receptor has a pentameric structure. Upon activation with nicotinic agonists the α7 receptor exhibited rapidly desensitizing cation currents that were blocked by nicotinic antagonists and showed inward rectification. The amplification of adrenal medulla RNA by reverse transcription polymerase chain reaction methods revealed an alternatively spliced isoform of the bovine α7 subunit, where the exon that codes for the M2 transmembrane segment was skipped during mRNA processing. Oocyte expression of this isoform does not yield functional channels. However, this alternative mRNA exhibits dose-dependent inhibition of α7 homomer expression when coinjected with the undeleted isoform.
    European Journal of Neuroscience 04/2006; 7(4):647 - 655. · 3.75 Impact Factor
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    ABSTRACT: 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.
    European Journal of Pharmacology 04/2006; 535(1-3):53-60. · 2.59 Impact Factor
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    ABSTRACT: 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.
    FEBS Letters 02/2006; 580(1):256-60. · 3.58 Impact Factor

Publication Stats

972 Citations
238.76 Total Impact Points

Institutions

  • 1998–2012
    • Universidad Miguel Hernández de Elche
      • • Instituto de Neurociencias
      • • Department of Pharmacology, Pediatrics and Organic Chemistry
      Elx, Valencia, Spain
  • 2006
    • Instituto de Neurociencias
      Alicante, Valencia, Spain
  • 1994–2006
    • University of Alicante
      • Departamento de Agroquímica y Bioquímica
      Alicante, Valencia, Spain
  • 2002
    • Spanish National Research Council
      Hispalis, Andalusia, Spain