Biomedical subjects

H R Arias

Publications and source records attributed to H R Arias.

At least 19 recordsLinked to original sources

PubMed2003

Lipid-protein interactions and effect of local anesthetics in acetylcholine receptor-rich membranes from Torpedo marmorata electric organ.

The selectivity of lipid-protein interaction for spin-labeled phospholipids and gangliosides in nicotinic acetylcholine receptor-rich membranes from Torpedo marmorata has been studied by ESR spectroscopy. The association constants of the spin-labeled lipids (relative to phosphatidylcholine) at pH 8.0 are in the order cardiolipin (5.1) approximately equal to stearic acid (4.9) approximately equal to phosphatidylinositol (4.7) > phosphatidylserine (2.7) > phosphatidylglycerol (1.7) > G(D1b) approximately equal to G(M1) approximately equal to G(M2) approximately equal to G(M3) approximately equal to phosphatidylcholine (1.0) > phosphatidylethanolamine (0.5). No selectivity for mono- or disialogangliosides is found over that for phosphatidylcholine. Aminated local anesthetics were found to compete with spin-labeled phosphatidylinositol, but to a much lesser extent with spin-labeled stearic acid, for sites on the intramembranous surface of the protein. The relative association constant of phosphatidylinositol was reduced in the presence of the different local anesthetics to the following extents: tetracaine (55%) > procaine (35%) approximately benzocaine (30%). For stearic acid, only tetracaine gave an appreciable reduction (30%) in association constant. These displacements represent an intrinsic difference in affinity of the local anesthetics for the lipid-protein interface because the membrane partition coefficients are in the order benzocaine >> tetracaine approximately procaine.

Anesthetics, Local↗

PubMed2001

Characterization of the dizocilpine binding site on the nicotinic acetylcholine receptor.

Although the dissociative anesthetic dizocilpine [(+)-MK-801] inhibits nicotinic acetylcholine receptor (AChR) function in a noncompetitive manner, the location of the dizocilpine binding site(s) has yet to be clearly established. Thus, to characterize the binding site for dizocilpine on the AChR we examined 1) the dissociation constant (K(d)) and stoichiometry of [(3)H]dizocilpine binding; 2) the displacement of dizocilpine radioligand binding by noncompetitive inhibitors (NCIs) and conversely dizocilpine displacement of fluorescent and radiolabeled NCIs from their respective high-affinity binding sites on the AChR; and 3) photoaffinity labeling of the AChR using (125)I-dizocilpine. The results establish that one high-affinity (K(d) = 4.8 microM) and several (3-6) low-affinity (K(d) = approximately 140 microM) binding sites exist for dizocilpine on the desensitized and resting AChR, respectively. The binding of the fluorescent NCIs ethidium, quinacrine, and crystal violet as well as [(3)H]thienylcyclohexylpiperidine was inhibited by dizocilpine on desensitized AChRs. However, Schild-type analyses indicate that only the inhibition of quinacrine in the desensitized state seems to be mediated by a mutually exclusive action. Photoaffinity labeling of the AChR by (125)I-dizocilpine was primarily restricted to the alpha1 subunit and subsequent mapping revealed that the principal sites of labeling are localized to the M4 (approximately 70%) and M1 (30%) transmembrane domains. Collectively, the data indicate that the high-affinity dizocilpine binding site is not located in the lumen of the ion channel but probably near the quinacrine binding locus at a nonluminal domain in the AChR desensitized state.

Animals↗

PubMed2001

Interaction of barbiturate analogs with the Torpedo californica nicotinic acetylcholine receptor ion channel.

Barbiturate-induced anesthesia is a complex mechanism that probably involves several ligand-gated ion channel superfamilies. One of these superfamilies includes the archetypical nicotinic acetylcholine receptor (nAChR), in which barbiturates act as noncompetitive antagonists. In this regard, we used the Torpedo californica nAChR and a series of barbiturate analogs to characterize the barbiturate binding site(s) on this superfamily member. [(14)C]Amobarbital binds to one high-affinity (K(d) = 3.7 microM) and several (approximately 11) low-affinity (K(d) = 930 microM) sites on the resting and desensitized nAChRs, respectively. Characteristics of the barbiturate binding site on the resting nAChR include: (1) a tight structure-activity relationship. For example, the barbiturate isobarbital [5-ethyl-5'-(2-methylbutyl) barbituric acid] is >10-fold less potent than its formula isomer amobarbital [5-ethyl-5'-(3-methylbutyl) barbituric acid] in inhibiting [(14)C]amobarbital binding. (2) A binding locus within the pore of the nAChR ion channel. Each of the barbiturate analogs inhibited the binding of [(3)H]tetracaine or photoincorporation of 3-trifluoromethyl-3-(m-[(125)I]iodophenyl) diazirine in a mutually exclusive manner. (3) Stereoselective binding. The R(+)-enantiomers of isobarbital and pentobarbital are approximately 2-fold more potent in inhibiting 3-trifluoromethyl-3-(m-[(125)I]iodophenyl) diazirine photoincorporation than the S(-)-enantiomers. Finally, molecular modeling suggests that within the channel, the pyrimidine ring of the barbiturate is located just above the highly conserved leucine ring (M2--9; e.g., delta Leu-265), whereas the 5' side chain projects downward, and depending upon its conformation, introduces steric hindrance to binding because of the restriction in the lumen of the channel introduced by the leucine side chains.

Amino Acid Sequence↗

PubMed2000

Localization of agonist and competitive antagonist binding sites on nicotinic acetylcholine receptors.

Identification of all residues involved in the recognition and binding of cholinergic ligands (e.g. agonists, competitive antagonists, and noncompetitive agonists) is a primary objective to understand which structural components are related to the physiological function of the nicotinic acetylcholine receptor (AChR). The picture for the localization of the agonist/competitive antagonist binding sites is now clearer in the light of newer and better experimental evidence. These sites are located mainly on both alpha subunits in a pocket approximately 30-35 A above the surface membrane. Since both alpha subunits are identical, the observed high and low affinity for different ligands on the receptor is conditioned by the interaction of the alpha subunit with other non-alpha subunits. This molecular interaction takes place at the interface formed by the different subunits. For example, the high-affinity acetylcholine (ACh) binding site of the muscle-type AChR is located on the alphadelta subunit interface, whereas the low-affinity ACh binding site is located on the alphagamma subunit interface. Regarding homomeric AChRs (e.g. alpha7, alpha8, and alpha9), up to five binding sites may be located on the alphaalpha subunit interfaces. From the point of view of subunit arrangement, the gamma subunit is in between both alpha subunits and the delta subunit follows the alpha aligned in a clockwise manner from the gamma. Although some competitive antagonists such as lophotoxin and alpha-bungarotoxin bind to the same high- and low-affinity sites as ACh, other cholinergic drugs may bind with opposite specificity. For instance, the location of the high- and the low-affinity binding site for curare-related drugs as well as for agonists such as the alkaloid nicotine and the potent analgesic epibatidine (only when the AChR is in the desensitized state) is determined by the alphagamma and the alphadelta subunit interface, respectively. The case of alpha-conotoxins (alpha-CoTxs) is unique since each alpha-CoTx from different species is recognized by a specific AChR type. In addition, the specificity of alpha-CoTxs for each subunit interface is species-dependent. In general terms we may state that both alpha subunits carry the principal component for the agonist/competitive antagonist binding sites, whereas the non-alpha subunits bear the complementary component. Concerning homomeric AChRs, both the principal and the complementary component exist on the alpha subunit. The principal component on the muscle-type AChR involves three loops-forming binding domains (loops A-C). Loop A (from mouse sequence) is mainly formed by residue Y(93), loop B is molded by amino acids W(149), Y(152), and probably G(153), while loop C is shaped by residues Y(190), C(192), C(193), and Y(198). The complementary component corresponding to each non-alpha subunit probably contributes with at least four loops. More specifically, the loops at the gamma subunit are: loop D which is formed by residue K(34), loop E that is designed by W(55) and E(57), loop F which is built by a stretch of amino acids comprising L(109), S(111), C(115), I(116), and Y(117), and finally loop G that is shaped by F(172) and by the negatively-charged amino acids D(174) and E(183). The complementary component on the delta subunit, which corresponds to the high-affinity ACh binding site, is formed by homologous loops. Regarding alpha-neurotoxins, several snake and alpha-CoTxs bear specific residues that are energetically coupled with their corresponding pairs on the AChR binding site. The principal component for snake alpha-neurotoxins is located on the residue sequence alpha1W(184)-D(200), which includes loop C. In addition, amino acid sequence 55-74 from the alpha1 subunit (which includes loop E), and residues gammaL(119) (close to loop F) and gammaE(176) (close to loop G) at the low-affinity binding site, or deltaL(121) (close to the homologous region of loop G) at the high-affinity binding site, are i

Animals↗

PubMed2000

Alpha-conotoxins.

alpha-Conotoxins (alpha-CgTxs) are a family of Cys-enriched peptides found in several marine snails from the genus Conus. These small peptides behave pharmacologically as competitive antagonists of the nicotinic acetylcholine receptor (AChR). The data indicate that (1) alpha-CgTxs are able to discriminate between muscle- and neuronal-type AChRs and even among distinct AChR subtypes; (2) the binding sites for alpha-CgTxs are located, like other cholinergic ligands, at the interface of alpha and non-alpha subunits (gamma, delta, and epsilon for the muscle-type AChR, and beta for several neuronal-type AChRs); (3) some alpha-CgTxs differentiate the high- from the low-affinity binding site found on either alpha/non-alpha subunit interface; and that (4) specific residues in the cholinergic binding site are energetically coupled with their corresponding pairs in the toxin stabilizing the alpha-CgTx-AChR complex. The alpha-CgTxs have proven to be excellent probes for studying the structure and function of the AChR family.

Amino Acid Sequence↗

PubMed1999

5-Doxylstearate-induced displacement of phencyclidine from its low-affinity binding sites on the nicotinic acetylcholine receptor.

Fatty acids as well as phencyclidine (PCP) inhibit the ion channel activity of the nicotinic acetylcholine receptor (AChR) by a noncompetitive mechanism. However, the exact localization of the fatty acid binding sites is unknown and, thus, the noncompetitive inhibitory mechanism for these endogenous modulators remains to be elucidated. In an attempt to determine the location of the fatty acid binding sites, we study the mutually exclusive action between 5-doxylstearate (5-SASL), a derivative of the endogenous noncompetitive antagonist (NCA) stearic acid, and other exogenous NCAs. For this purpose, both equilibrium and competitive binding assays using fluorescent and radiolabeled ligands were performed on desensitized AChRs. More specifically, we determined: (i) the effect of 5-SASL on the binding of the exogenous NCA [(3)H]PCP; (ii) the effect of 5-SASL on the binding of either quinacrine or ethidium, two fluorescent NCAs from exogenous origin; and (iii) the PCP-induced displacement of quinacrine and ethidium from their respective high-affinity binding sites. Our first target (i) is carried out by measuring the [(3)H]PCP binding in the absence or in the presence of increasing concentrations of 5-SASL. We found that 5-SASL displaces PCP from its low-affinity binding sites. The low-affinity PCP binding sites were pharmacologically characterized by an apparent dissociation constant (K(d)) of 6.1 +/- 5.0 microM and a stoichiometry of 3.7 +/- 1.5 sites per AChR. The fact that 5-SASL increased the apparent K(d) without changing the number of sites per AChR is indicative of a mutually exclusive action. From these results, an apparent inhibition constant (K(i)) of 75 +/- 31 microM for 5-SASL was calculated. In addition, 5-SASL affected neither the apparent K(d) (0.46 +/- 0.37 microM) nor the stoichiometry (1.07 +/- 0.57 sites per AChR) of the high-affinity PCP binding site. The second objective (ii) is achieved by titrating either quinacrine or ethidium into AChR native membranes in the absence or in the presence of increasing concentrations of 5-SASL. These experiments showed that 5-SASL efficiently increased the apparent K(d) of quinacrine without perturbing the interaction of ethidium with its high-affinity locus. Considering that (a) 5-SASL effectively quenched the AChR-bound quinacrine fluorescence (H. R. Arias, Biochim. Biophys. Acta 1347, 9-22, 1997) and (b) fluorescence-quenching is a short-range process, it is possible to suggest that 5-SASL displaces quinacrine from its high-affinity binding site by a steric mechanism. In this regard, a K(i) of 38 +/- 5 microM for 5-SASL was calculated. Concerning the last objective (iii), AChR-bound quinacrine or ethidium was back titrated with PCP. Two PCP K(i) values were obtained by fitting the displacement plots by nonlinear regression with two components. The lowest K(i) values obtained for either quinacrine (0.86 +/- 0.37 microM) or ethidium (0. 29 +/- 0.23 microM) displacement from their respective high-affinity binding sites coincide with the previously determined high-affinity [(3)H]PCP K(d). In addition, the highest K(i) values obtained for either NCA displacement are in the same concentration range as the observed low-affinity [(3)H]PCP K(d). Taking into account all experimental data, we reached the following conclusions: (i) fatty acid molecules, or at least 5-SASL, sterically interact with both the PCP low-affinity and the quinacrine high-affinity binding sites; (ii) the low-affinity PCP binding sites, as well as the high-affinity quinacrine locus, are located at the nonannular lipid domain of the AChR; and, finally, (iii) fatty acid molecules are not accessible to the lumen of the ion channel, indicating an allosteric mode of action for fatty acids to inhibit ion flux. Thus, the 5-SASL, the quinacrine high-affinity, and the PCP low-affinity binding sites are all located at overlapping nonannular loci on the muscle-type AChR.

Animals↗

PubMed1999

Role of local anesthetics on both cholinergic and serotonergic ionotropic receptors.

A great body of experimental evidence indicates that the main target for the pharmacological action of local anesthetics (LAs) is the voltage-gated Na+ channel. However, the epidural and spinal anesthesia as well as the behavioral effects of LAs cannot be explained exclusively by its inhibitory effect on the voltage-gated Na+ channel. Thus, the involvement of other ion channel receptors has been suggested. Particularly, two members of the neurotransmitter-gated ion channel receptor superfamily, the nicotinic acetylcholine receptor (AChR) and the 5-hydroxytryptamine receptor (5-HT3R type). In this regard, the aim of this review is to explain and delineate the mechanism by which LAs inhibit both ionotropic receptors from peripheral and central nervous systems. Local anesthetics inhibit the ion channel activity of both muscle- and neuronal-type AChRs in a noncompetitive fashion. Additionally, LAs inhibit the 5-HT3R by competing with the serotonergic agonist binding sites. The noncompetitive inhibitory action of LAs on the AChR is ascribed to two possible blocking mechanisms. An open-channel-blocking mechanism where the drug binds to the open channel and/or an allosteric mechanism where LAs bind to closed channels. The open-channel-blocking mechanism is in accord with the existence of high-affinity LA binding sites located in the ion channel. The allosteric mechanism seems to be physiologically more relevant than the open-channel-blocking mechanism. The inhibitory property of LAs is also elicited by binding to several low-affinity sites positioned at the lipid-AChR interface. However, there is no clearcut evidence indicating whether these sites are located at either the annular or the nonannular lipid domain. Both tertiary (protonated) and quaternary LAs gain the interior of the channel through the hydrophilic pathway formed by the extracellular ion channel's mouth with the concomitant ion flux blockade. Nevertheless, an alternative mode of action is proposed for both deprotonated tertiary and permanently-uncharged LAs: they may pass from the lipid membrane core to the lumen of the ion channel through a hydrophobic pathway. Perhaps this hydrophobic pathway is structurally related to the nonannular lipid domain. Regarding the LA binding site location on the 5-HT3R, at least two amino acids have been involved. Glutamic acid at position 106 which is located in a residue sequence homologous to loop A from the principal component of the binding site for cholinergic agonists and competitive antagonists, and Trp67 which is positioned in a stretch of amino acids homologous to loop F from the complementary component of the cholinergic ligand binding site.

Anesthetics, Local↗

PubMed1998

Binding sites for exogenous and endogenous non-competitive inhibitors of the nicotinic acetylcholine receptor.

The nicotinic acetylcholine receptor (AChR) is the paradigm of the neurotransmitter-gated ion channel superfamily. The pharmacological behavior of the AChR can be described as three basic processes that progress sequentially. First, the neurotransmitter acetylcholine (ACh) binds the receptor. Next, the intrinsically coupled ion channel opens upon ACh binding with subsequent ion flux activity. Finally, the AChR becomes desensitized, a process where the ion channel becomes closed in the prolonged presence of ACh. The existing equilibrium among these physiologically relevant processes can be perturbed by the pharmacological action of different drugs. In particular, non-competitive inhibitors (NCIs) inhibit the ion flux and enhance the desensitization rate of the AChR. The action of NCIs was studied using several drugs of exogenous origin. These include compounds such as chlorpromazine (CPZ), triphenylmethylphosphonium (TPMP+), the local anesthetics QX-222 and meproadifen, trifluoromethyl-iodophenyldiazirine (TID), phencyclidine (PCP), histrionicotoxin (HTX), quinacrine, and ethidium. In order to understand the mechanism by which NCIs exert their pharmacological properties several laboratories have studied the structural characteristics of their binding sites, including their respective locations on the receptor. One of the main objectives of this review is to discuss all available experimental evidence regarding the specific localization of the binding sites for exogenous NCIs. For example, it is known that the so-called luminal NCIs bind to a series of ring-forming amino acids in the ion channel. Particularly CPZ, TPMP+, QX-222, cembranoids, and PCP bind to the serine, the threonine, and the leucine ring, whereas TID and meproadifen bind to the valine and extracellular rings, respectively. On the other hand, quinacrine and ethidium, termed non-luminal NCIs, bind to sites outside the channel lumen. Specifically, quinacrine binds to a non-annular lipid domain located approximately 7 A from the lipid-water interface and ethidium binds to the vestibule of the AChR in a site located approximately 46 A away from the membrane surface and equidistant from both ACh binding sites. The non-annular lipid domain has been suggested to be located at the intermolecular interfaces of the five AChR subunits and/or at the interstices of the four (M1-M4) transmembrane domains. One of the most important concepts in neurochemistry is that receptor proteins can be modulated by endogenous substances other than their specific agonists. Among membrane-embedded receptors, the AChR is one of the best examples of this behavior. In this regard, the AChR is non-competitively modulated by diverse molecules such as lipids (fatty acids and steroids), the neuropeptide substance P, and the neurotransmitter 5-hydroxytryptamine (5-HT). It is important to take into account that the above mentioned modulation is produced through a direct binding of these endogenous molecules to the AChR. Since this is a physiologically relevant issue, it is useful to elucidate the structural components of the binding site for each endogenous NCI. In this regard, another important aim of this work is to review all available information related to the specific localization of the binding sites for endogenous NCIs. For example, it is known that both neurotransmitters substance P and 5-HT bind to the lumen of the ion channel. Particularly, the locus for substance P is found in the deltaM2 domain, whereas the binding site for 5-HT and related compounds is putatively located on both the serine and the threonine ring. Instead, fatty acid and steroid molecules bind to non-luminal sites. More specifically, fatty acids may bind to the belt surrounding the intramembranous perimeter of the AChR, namely the annular lipid domain, and/or to the high-affinity quinacrine site which is located at a non-annular lipid domain. Additionally, steroids may bind to a site located on the extracellular hydrophi

Animals↗

PubMed1998

Noncompetitive inhibition of nicotinic acetylcholine receptors by endogenous molecules.

Over the past few decades much effort has been expended elucidating the key domains of the nicotinic acetylcholine receptor (AChR) responsible for agonist binding, ion conduction, and gating. An emerging concept in the receptor field has been to consider the receptor entity as a signal transducer that suffers modulatory control by allosterically acting ligands. Of particular interest are the molecules that inhibit the agonist-evoked ion flux activity in a noncompetitive manner: the so-called noncompetitive inhibitors (NCIs). The actual knowledge on the action of NCIs was obtained by using several drugs from exogenous origin. However, several lines of investigation indicate that the receptor protein can be modulated by endogenous substances other than acetylcholine. In this regard, we outline the progress evidenced on the localization of binding sites for drugs of endogenous origin that have been found to directly interact with the AChR in a noncompetitive fashion. Among them we can quote lipids such as steroids and fatty acids, the neurotransmitter 5-hydroxytryptamine (5-HT) and related compounds, as well as the neuropeptide substance P. We present the available experimental evidence indicating the existence of both luminal (located into the ion channel) and nonluminal (located out of the ion channel) binding sites for endogenous NCIs. Particularly, the binding site for substance P is found in the delta M2 domain. In addition, the locus for 5-HT is putatively located in the ion channel close to the serine ring, whereas the binding site for two competitive antagonists of 5-HT receptors (e.g., methysergide and spiperone) is located closer to the external end of the ion channel. Instead, fatty acid and steroid molecules bind to nonluminal sites. More specifically, fatty acids may bind to the annular lipid domain of the AChR or/and to the high-affinity quinacrine site (a NCI from exogenous origin) which is located at a nonannular lipid domain. Additionally, steroids may bind to a site located on the extracellular hydrophilic domain of the AChR or/and at the lipid-protein interface, specifically, at the annular lipid domain and/or close to the nonannular quinacrine binding site.

Animals↗

PubMed1997

The high-affinity quinacrine binding site is located at a non-annular lipid domain of the nicotinic acetylcholine receptor.

This work deals with the localization of the high-affinity non-competitive quinacrine binding site on the muscle-type nicotinic acetylcholine receptor (AChR). Specifically, quantitative steady-state fluorescence spectroscopy is used to determine whether quinacrine binds to a site located at either the annular or the non-annular lipid domain. For this purpose, we measure the ability of spin-labelled phosphatidylcholine (SL-PC) to quench AChR-bound quinacrine, AChR-bound ethidium and membrane-partitioned 7-(9-anthroyloxy)stearate (7-AS) fluorescence. Additionally, we compare the accessibility of SL-PC which is considered to bind only to the annular lipid domain of the AChR with the accessibility of two non-annular domain-sensing lipids such as 5-doxylstearate (5-SAL) and spin-labelled androstane (ASL). Initial experiments using 7-AS established the experimental conditions for maximum SL-PC membrane partitioning. The non-specific quenching elicited by increasing turbidity of the sample after addition of SL-PC is corrected by means of parallel experiments with unlabelled egg yolk phosphatidylcholine. After correction, the SL-PC quenching experiments show the following order in quenching efficiency: 7-AS > quinacrine >> ethidium. The relative intrinsic sensitivity of quinacrine to TEMPO paramagnetic quenching in acetonitrile is considered to be approximately two times higher than that for 7-AS. Thus, SL-PC was found to be more accessible (about 5-fold) to the membrane-partitioned 7-AS than to the quinacrine locus. In addition, SL-PC was virtually not accessible to the high-affinity non-luminal binding site for ethidium. The relative capacity of SL-PC, 5-SAL, and ASL to quench AChR-bound quinacrine fluorescence indicated that the spin-labelled lipid accessibility to the quinacrine binding site follows the order: 5-SAL > ASL >> SL-PC. Examination of the effect of high concentrations of 5-SAL, of its unlabelled parent stearate, and of SL-PC on ethidium and quinacrine binding showed that: (a) both fatty acids displace quinacrine, but not ethidium, from its high-affinity binding site, however (b) 5-SAL was found to be more effective than stearate to displace quinacrine from its locus, whereas (c) SL-PC competes neither for the ethidium locus nor for the quinacrine binding site. The results suggest that the high-affinity binding site for quinacrine is located at a non-annular lipid domain of the AChR. This particular area has been considered to be located at the intramolecular interfaces of the five AChR subunits and/or at the interstices of the transmembrane domains.

Animals↗

PubMed1997

Dramatic saccharide-mediated protection of chaotropic-induced deactivation of concanavalin A.

This work provides evidence of a physical instance in which some proteins that are usually inactivated under strong chaotropic conditions may become fully resistant through the occupancy of their binding sites with suitable ligands. In this regard, we found that Moluccella laevis lectin remains stable in the presence of denaturant concentrations of urea when an appropriate saccharide is bound to the protein (Alperin, D.M., Latter, H., Lis, H., and Sharon, N. (1992) Biochem. J. 285, 1-4). Extending this finding, we now demonstrate that the occupancy of the ligand binding sites of concanavalin A (Con A) with appropriate carbohydrates completely prevents the denaturation course elicited by 8 M urea at pH 7.4. In addition, the protecting efficiency of the saccharides was shown to be directly related to their specificities for the lectin. The observed saccharide protection follows the order:methyl alpha-D-mannopyranoside > methyl alpha-D-glucopyr-anoside > mannose > fructose > glucose. Concomitantly, the active tetrameric lectin with a molecular mass of approximately 105 kDa is preserved in 8 M urea when methyl alpha-D-mannopyranoside (100 mM) is present in the medium.

Allosteric Regulation↗

PubMed1997

Topology of ligand binding sites on the nicotinic acetylcholine receptor.

The nicotinic acetylcholine receptor (AChR) presents two very well differentiated domains for ligand binding that account for different cholinergic properties. In the hydrophilic extracellular region of both alpha subunits there exist the binding sites for agonists such as the neurotransmitter acetylcholine (ACh) and for competitive antagonists such as d-tubocurarine. Agonists trigger the channel opening upon binding while competitive antagonists compete for the former ones and inhibit its pharmacological action. Identification of all residues involved in recognition and binding of agonist and competitive antagonists is a primary objective in order to understand which structural components are related to the physiological function of the AChR. The picture for the localisation of the agonist/competitive antagonist binding sites is now clearer in the light of newer and better experimental evidence. These sites are mainly located on both alpha subunits in a pocket approximately 30-35 A above the surface membrane. Since both alpha subunits are sequentially identical, the observed high and low affinity for agonists on the receptor is conditioned by the interaction of the alpha subunit with the delta or the gamma chain, respectively. This relationship is opposite for curare-related drugs. This molecular interaction takes place probably at the interface formed by the different subunits. The principal component for the agonist/competitive antagonist binding sites involves several aromatic residues, in addition to the cysteine pair at 192-193, in three loops-forming binding domains (loops A-C). Other residues such as the negatively changed aspartates and glutamates (loop D), Thr or Tyr (loop E), and Trp (loop F) from non-alpha subunits were also found to form the complementary component of the agonist/competitive antagonist binding sites. Neurotoxins such as alpha-, kappa-bungarotoxin and several alpha-conotoxins seem to partially overlap with the agonist/competitive antagonist binding sites at multiple point of contacts. The alpha subunits also carry the binding site for certain acetylcholinesterase inhibitors such as eserine and for the neurotransmitter 5-hydroxytryptamine which activate the receptor without interacting with the classical agonist binding sites. The link between specific subunits by means of the binding of ACh molecules might play a pivotal role in the relative shift among receptor subunits. This conformational change would allow for the opening of the intrinsic receptor cation channel transducting the external chemical signal elicited by the agonist into membrane depolarisation. The ion flux activity can be inhibited by non-competitive inhibitors (NCIs). For this kind of drugs, a population of low-affinity binding sites has been found at the lipid-protein interface of the AChR. In addition, several high-affinity binding sites have been found to be located at different rings on the M2 transmembrane domain, namely luminal binding sites. In this regard, the serine ring is the locus for exogenous NCIs such as chlorpromazine, triphenylmethylphosphonium, the local anaesthetic QX-222, phencyclidine, and trifluoromethyliodophenyldiazirine. Trifluoromethyliodophenyldiazirine also binds to the valine ring, which is the postulated site for cembranoids. Additionally, the local anaesthetic meproadifen binding site seems to be located at the outer or extracellular ring. Interestingly, the M2 domain is also the locus for endogenous NCIs such as the neuropeptide substance P and the neurotransmitter 5-hydroxytryptamine. In contrast with this fact, experimental evidence supports the hypothesis for the existence of other NCI high-affinity binding sites located not at the channel lumen but at non-luminal binding domains. (ABSTRACT TRUNCATED)

Acetylcholine↗

PubMed1996

Temperature and ionic strength dependence of quinacrine binding and quinacrine displacement elicited by high concentrations of agonists on the nicotinic acetylcholine receptor.

This paper displays an attempt to elucidate the inhibitory mechanism of the nicotinic acetylcholine receptor (AChR) by spectroscopic means. Specifically, quantitative fluorescence spectroscopy was used to characterize: (1) the mechanism of quinacrine binding to its high-affinity noncompetitive inhibitor site located at the lipid-protein interface of the AChR and (2) the process by which agonists at high concentrations sterically compete for the quinacrine locus. For the first purpose, we study the temperature and ionic strength dependence of quinacrine binding by measuring the apparent dissociation constant (Kd) of quinacrine at the temperature range of 4-23 degrees C and in the sodium chloride (NaCl) concentration order of 0-250 mM. For the second objective, AChR native membranes from Torpedo californica electric organ suspended in buffer 10 mM sodium phosphate, pH 7.4, were preincubated with quinacrine for 2 h in the presence or in the absence of phencyclidine (PCP). Then, the PCP-sensitive quinacrine fluorescence was monitored while high concentrations of cholinergic agonists such as suberyldicholine, acetylcholine (ACh), or carbamylcholine were added to the suspension. By repeating these agonist back titrations at 4, 9, and 15 degrees C in the absence of NaCl and at 4 degrees C in the presence of 100 mM NaCl, we determined the temperature and ionic strength dependence of agonist binding to the quinacrine domain. These experiments suggest that the binding of both quinacrine (measured in the temperature range from 15 to 23 degrees C) and agonists at high concentrations (measured in the temperature regime of 4-15 degrees C) are enthalpy-driven processes, albeit that quinacrine binding is exothermic and agonist binding is endothermic. One plausible model to explain our results is that the quinacrine molecule needs first to be sterically well oriented to further enter into its binding site located in a crevice at the lipid-protein interface, whereas agonist molecules do not. Additionally, a relatively minimal electrostatic component is present in the quinacrine locus. Interestingly, the agonist inhibition constant values determined at 4 degrees C in the presence of 100 mM NaCl showed an exact correlation (slope = 1.03) with the reported concentration values of agonist that inhibit 50% of the maximum 86Rb+ efflux from AChR native vesicles in a 10-s assay with 80-85% of the alpha-bungarotoxin AChR sites occupied at zero membrane potential [S. A. Forman, L. L. Firestone, and K. W. Miller (1987) Biochemistry 26, 2807-2814]. This interdependence strongly supports the existence of a structural relationship between the agonist self-inhibitory binding site and the quinacrine locus. Although there exist evidence indicating that the process of agonist self-inhibition is mediated by a steric blockage of the ion channel, the occurrence of an agonist self-inhibitory binding site not located in the lumen channel indicates an allosteric mechanism for ion channel inhibition.

Animals↗

PubMed1996

Agonist self-inhibitory binding site of the nicotinic acetylcholine receptor.

A major focus of current research on the nicotinic acetylcholine receptor (AChR) has been to understand the molecular mechanism of ion channel inhibition. In particular, we put special emphasis on the description of the localization of the agonist self-inhibitory binding site. Binding of agonist in the millimolar concentration range to this particular site produces inhibition of the ion flux activity previously elicited by the same agonist at micromolar concentrations. Due to the similitude in the pharmacological and electrophysiological behavior in inhibiting the ion channel of both high agonist concentrations and noncompetitive antagonists, we first describe the localization of noncompetitive inhibitor binding sites on the AChR. There is a great body of experimental evidence for the existence and location of luminal high-affinity noncompetitive inhibitor binding sites. In this regard, the most simple mechanism to describe the action of noncompetitive inhibitors which bind to luminal sites and, by its semblance, the agonist self-inhibition itself, is based on the assumption that these compounds enter the open channel, bind to different rings within the M2 transmembrane domain of the receptor, and block cation flux by occluding the receptor pore. However, the existence of high-affinity nonluminal noncompetitive inhibitor binding sites is not consistent with the open-channel-blocking mechanism. Instead, the presence of the quinacrine locus at the lipid-protein (alpha M1) interface approximately 7 A from the lipid-water interface and the ethidium domain located approximately 46 A from the membrane surface in the wall of the vestibule open the possibility for the regulation of cation permeation by an allosteric process. Additionally, the observed (at least partially) overlapping between the quinacrine and the agonist self-inhibitory binding site also suggests an allosteric process for agonist self-inhibition. For this alternative mechanism, cholinergic agonist molecules first need to be partitioned into (or to be adsorbed onto) the lipid membrane to further interact with its binding site located at the lipid-protein interface.

Animals↗

PubMed1995

Differential agonist-induced displacement of quinacrine and ethidium from their respective histrionicotoxin-sensitive binding sites on the Torpedo acetylcholine receptor.

Fluorescence spectroscopy was used to begin to localize the agonist inhibitory binding site on the nicotinic acetylcholine receptor (AcChR) from Torpedo californica. High concentrations of three cholinergic agonists, suberyldicholine (SubCh), acetylcholine (AcCh), and carbamylcholine (CCh), differentially inhibited the binding of two noncompetitive inhibitors (NCIs), quinacrine and ethidium, which bind at distinctly different loci on the desensitized AcChR at zero membrane potential. The agonist-induced inhibition of quinacrine binding occurred at significantly lower (17-fold) concentrations than the inhibition of ethidium binding. Schild plots of SubCh inhibition of ethidium and quinacrine binding showed the competitive nature of the agonist inhibition of the binding of these two NCIs. The quenching constants for short-range quenching of receptor-bound quinacrine and ethidium fluorescence by spin-labeled acetylcholine were about the same as their inhibition constants for agonist-induced displacement of AcChR-bound quinacrine and ethidium. The results demonstrate that agonists can directly bind to both the quinacrine and the ethidium binding sites, albeit at different agonist concentrations. Because the agonist-induced displacement of receptor-bound quinacrine occurs at significantly lower concentrations than the displacement of ethidium, the quinacrine binding site is more likely than the ethidium binding site to form part of the agonist inhibitory binding site.

Acetylcholine↗

PubMed1994

Antibody-induced conformational changes in the Torpedo nicotinic acetylcholine receptor: a fluorescence study.

Quantitative fluorescence spectroscopy was used to develop a structural picture of the effects of two monoclonal antibodies (mAbs) on the conformation of the Torpedo nicotinic acetylcholine receptor (nAChR). The two mAbs (A6 and B1) examined selectively blocked ligand binding to either the high-affinity (A) or the low-affinity (B) binding sites for agonists/competitive antagonists. The distances between dansyl-C6-choline bound to the unblocked agonist/competitive antagonist binding site and one of two lipophilic probes (C12-eosin or C18-rhodamine) partitioned into the lipid membrane were estimated by using fluorescence resonance energy transfer. Control experiments demonstrated that both mAbs decreased the affinity and fluorescence lifetime of receptor-bound dansyl-C6-choline. The binding of the B1 mAb to the B site did not significantly change the calculated distance between the unblocked A binding site and the membrane surface. However, the binding of the A6 mAb to the A site induced the B site to move into close proximity to the lipid membrane. This conformational change was confirmed by a 45-fold increase in the paramagnetic quenching of the B-site-bound dansyl-C6-choline fluorescence by lipid-intercalated 5-doxylstearate. The results indicate that these mAbs not only selectively block ligand binding to either the A or the B acetylcholine sites but also, in the case of the A6 mAb, induce global conformational changes of the receptor, which appear to involve a movement of the B binding site into close proximity of the lipid membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Interaction of merocyanine 540 with nicotinic acetylcholine receptor membranes from Discopyge tschudii electric organ.

Interactions between merocyanine 540 (MC540) and nicotinic acetylcholine receptor (AChR) have been studied by visible absorption spectroscopy using native receptor-rich membranes from Discopyge tschudii electric tissue and liposomes obtained by aqueous dispersion of endogenous lipids extracted from the same tissue. The fact that merocyanine partitions into the membrane when this is in the liquid-crystalline state, exhibiting a characteristic peak at 567 nm, was exploited to obtain quantitative information about the physical state of the AChR-rich membrane. Spectra of MC540 revealed that this molecule was preferentially incorporated into AChR-rich membranes, with an affinity (Kdapp 30 microM) 10-fold higher than that in liposomes (Kdapp 290 microM). Changes were observed in the equilibrium dissociation constant of MC540 at different temperatures: the two-fold higher affinity at 8 degrees C than at 23 degrees C can be rationalized in terms of a higher value of the overall dimerization constant (Kdim) at the lower temperature. The local anaesthetic benzocaine competed for MC540 binding sites with higher potency in AChR-rich native membranes than in liposomes made with endogenous lipids. This competition was found to be AChR concentration-dependent, whereas in liposomes the displacement was constant at different lipid/MC540 molar ratios. Titration experiments yielded an apparent dissociation constant for benzocaine of 0.6 mM and 0.7 mM for liposomes and AChR-rich membranes, respectively. The possible location of the benzocaine binding site is deduced from the competition experiments to be at the lipid annulus surrounding the nicotinic AChR protein.

Animals↗

PubMed1993

Quinacrine and ethidium bind to different loci on the Torpedo acetylcholine receptor.

Fluorescence spectroscopy was used to determine whether quinacrine and ethidium, two high-affinity noncompetitive inhibitors of the Torpedo acetylcholine receptor (AcChR), bind to the same loci. The ability of three nitroxide spin-labels, 5-doxylstearate (5-SAL), spin-labeled androstane (ASL), and TEMPO, to quench receptor-bound quinacrine and ethidium fluorescence was measured. When bound to a phencyclidine-displaceable site on the AcChR, quinacrine was 16.9 and 19 times more efficiently quenched than ethidium by the highly lipophilic 5-SAL and ASL, respectively. TEMPO, which has a limited ability to partition into Torpedo plasma membranes (< 1%), was only twice as efficient at quenching receptor-bound quinacrine than ethidium fluorescence. The relative sensitivity of quinacrine and ethidium fluorescence to paramagnetic quenching was examined in three solvents, 1-butanol, sodium phosphate buffer, and acetonitrile, with TEMPO as a quencher. The results from the different solvents demonstrate that quinacrine fluorescence is intrinsically 1.4-3.6 times more sensitive than ethidium fluorescence to quenching by nitroxide spin-labels. Examination of the effect of high concentrations of 5-SAL on ethidium and quinacrine dissociation constants showed that quinacrine but not ethidium binding was competitively inhibited. Together, these results indicate that although quinacrine and ethidium bind in a mutually exclusive manner, the two inhibitors interact at different loci on the AcChR. Whereas the ethidium binding site is at a distance from membrane lipids, probably in or near the lumen, the quinacrine binding site appears to be at a lipid-protein interface in the transmembrane domain and at a distance from the lumen.

Animals↗