Paradigmatic obstacles to improving the health of populations--implications for health policy

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.     

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     

Formation of the nicotinic acetylcholine receptor binding sites

Nicotinic acetylcholine receptors (AChRs) are activated by ACh binding to two sites located on different alpha subunits. The two alpha subunits, alpha gamma and alpha delta, are distinguished by their interface with gamma and delta subunits. We have characterized the formation of the ACh binding sites and found, contrary to the current model, that the sites form at different times and in a set order. The first site forms on alpha gamma subunits during the process of subunit assembly. Our data are consistent with the appearance of this site on alpha beta gamma delta subunit tetramers soon after the site for the competitive antagonist alpha-bungarotoxin has formed and delta subunits have assembled with alpha beta gamma trimers. The second site is located on alpha delta subunits and forms after AChR subunits have assembled into alpha2 beta gamma delta pentamers. By determining the order in which the ACh binding sites form, we have also identified the sites in which the delta and second alpha subunits associate during subunit assembly.     

Separate agonist and peptide antagonist binding sites of the oxytocin receptor defined by their transfer into the V2 vasopressin receptor

The neurohypophyseal nonapeptide oxytocin (OT) is the main hormone responsible for the initiation of labor; uterus contraction can be enhanced by application of oxytocin or suppressed by oxytocin antagonists. By transfer of domains from the G protein-coupled OT receptor into the related V2 vasopressin receptor, chimeric "gain in function" V2/OT receptors were produced that were able to bind either OT receptor agonists or a competitive peptide antagonist with high affinity. The binding site for the OT antagonist d(CH2)5[Tyr(Me)2,Thr4,Orn8,Tyr9]vasotocin was found to be formed by transmembrane helices 1, 2, and 7 with a major contribution to binding affinity by the upper part of helix 7. These transmembrane receptor regions could be excluded from participating in OT binding. For agonist binding and selectivity the first three extracellular receptor domains were most important. The interaction of the N-terminal domain and of the first extracellular loop of the OT receptor with the linear C-terminal tripeptidic part of oxytocin was demonstrated. Furthermore, the second extracellular loop of the OT receptor could be identified to interact with the cyclic hormone part. These three domains contribute to OT binding by synergistic interaction with oxytocin but not with the competitive antagonist. Our results provide evidence for the existence of separate domains and different conformations of a peptide hormone receptor involved in binding and selectivity for agonists and peptide antagonists.     

Requirements for binding and signaling of the kinase domain receptor for vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is a dimeric hormone that controls much of vascular development through binding and activation of its kinase domain receptor (KDR). We produced analogs of VEGF that show it has two receptor-binding sites which are located near the poles of the dimer and straddle the interface between subunits. Deletion experiments in KDR indicate that of the seven IgG-like domains in the extracellular domain, only domains 2-3 are needed for tight binding of VEGF. Monomeric forms of the extracellular domain of KDR bind approximately 100 times weaker than dimeric forms showing a strong avidity component for binding of VEGF to predimerized forms of the receptor. Based upon these structure-function studies and a mechanism in which receptor dimerization is critical for signaling, we constructed a receptor antagonist in the form of a heterodimer of VEGF that contained one functional and one non-functional site. These studies establish a functional foundation for the design of VEGF analogs, mimics, and antagonists.     

Mutation in the M1 domain of the acetylcholine receptor alpha subunit decreases the rate of agonist dissociation

We describe the kinetic consequences of the mutation N217K in the M1 domain of the acetylcholine receptor (AChR) alpha subunit that causes a slow channel congenital myasthenic syndrome (SCCMS). We previously showed that receptors containing alpha N217K expressed in 293 HEK cells open in prolonged activation episodes strikingly similar to those observed at the SCCMS end plates. Here we use single channel kinetic analysis to show that the prolonged activation episodes result primarily from slowing of the rate of acetylcholine (ACh) dissociation from the binding site. Rate constants for channel opening and closing are also slowed but to much smaller extents. The rate constants derived from kinetic analysis also describe the concentration dependence of receptor activation, revealing a 20-fold shift in the EC50 to lower agonist concentrations for alpha N217K. The apparent affinity of ACh binding, measured by competition against the rate of 125I-alpha-bungarotoxin binding, is also enhanced 20-fold by alpha N217K. Both the slowing of ACh dissociation and enhanced apparent affinity are specific to the lysine substitution, as the glutamine and glutamate substitutions have no effect. Substituting lysine for the equivalent asparagine in the beta, epsilon, or delta subunits does not affect the kinetics of receptor activation or apparent agonist affinity. The results show that a mutation in the amino-terminal portion of the M1 domain produces a localized perturbation that stabilizes agonist bound to the resting state of the AChR.     

Differential coupling of rat D2 dopamine receptor isoforms expressed in Spodoptera frugiperda insect cells

By using a baculovirus expression system, the two isoforms of the rat D2 dopamine receptor were expressed at densities ranging up to 15 pmol/mg of protein. D2L and D2S dopamine receptors expressed in aline of Spodoptera frugiperda (Sf9) insect cells Sf9cells, displayed high affinity for the antagonists spiroperidol and (+)-butaclamol and the agonist N-propylnorapomorphine. Antisera raised against the D2 receptor immunoprecipitated binding sites for a radiolabeled D2 antagonist from solubilized extracts of infected Sf9cells. In immunoblots of Sf9cells infected with recombinant D2 baculovirus, these antisera recognized a major species of protein of approximately 46 kDa. Photoaffinity-labeling of infected Sf9cells using N-(p-azido-m-[125I]iodophenethyl)spiperone also identified a protein of this size, suggesting that D2 receptors expressed in Sf9cells are largely unglycosylated. In cells expressing receptors at a density greater than 1 pmol/mg, GTP-sensitive, high-affinity binding of agonists was not detected in studies of the inhibition of the binding of a radiolabeled D2 antagonist. When expression levels were under 1 pmol/mg, the binding of agonists was sensitive to the addition of guanine nucleotides, indicating that D2 receptors were coupled to endogenous G proteins. Endogenous G proteins enable both isoforms of D2 receptors to couple to the inhibition of adenylyl cyclase activity. The high-affinity state of the D2 receptor was directly measured using a radiolabeled agonist. Although the density of receptors increased with longer times after infection, the density of high-affinity sites reached a maximum of approximately 40 fmol/mg 30 to 36 hr after infection. Coexpression of D2 receptors and G protein subunits in Sf9cells dramatically increased the density of high-affinity sites, whereas the total density of receptors was unchanged, confirming that D2 receptors in Sf9 cells can exist in the high-affinity-coupled state, but that appropriate G proteins are expressed at relatively low levels. The density of D2S receptors converted to a coupled, agonist-preferring state when coexpressed with G proteins subunits (alpha i1, beta 1 and gamma 2) was 5 times greater than that of D2L receptors expressed under the same conditions, consistent with the hypothesis that D2 dopamine receptor isoforms differentially couple to alpha i1.     

Split-receptors in the tachykinin neurokinin-1 system--mutational analysis of intracellular loop 3

Several membrane proteins have been functionally expressed from non-covalently coupled, contiguous segments especially with the split-site located between natural domains. Experiments using such 'split-proteins' were here performed in the tachykinin neurokinin-1 (NK1) receptor with co-expression of contiguous segments with split-sites positioned in various intracellular and extracellular loops. The construct where the split-site was located in intracellular loop 3 gave a reasonable expression level of substance-P-binding sites, i.e. 12% of wild-type expression. Of the other split-receptors tested, only the one with the split-site located just outside transmembrane (TM) segment-V gave any detectable substance P binding, which however only was 1% of the wild-type expression level. The construct with the split-site located in intracellular loop 3 bound all of the tested peptide agonists and non-peptide antagonists with normal affinity and was able to stimulate inositol phosphate turnover with a normal EC50 for substance P and an Emax according to the expression level. When intracellular loop 3 was either extended with 112 amino acid residues derived from the muscarine M2 receptor or, when major parts of the loop were deleted in the non-split NK1 receptor, the affinity for neither substance P nor for the prototype nonpeptide antagonist, CP96,345 was affected, yet an increase in EC50 for substance P was observed. Also in the split-receptor, most of intracellular loop 3 could be substituted or even deleted without affecting ligand affinity, although a decreased expression level was observed in constructs having major deletions. It is concluded, that the NK1 receptor is preferentially reconstituted by co-expression of a putative A-domain including TM-I-V and a B-domain including TM-VI and -VII. It is suggested that a number of rhodopsin-like 7TM receptors may function as two-domain structures based on the finding that a network of short loops has been highly conserved within each of the putative domains and, that these domains are separated by a relatively long and in respect of length poorly conserved loop, i.e. intracellular loop 3.     

Congenital myasthenic syndromes: clinical and genetic analysis of 18 patients

Congenital myasthenic syndromes (CMS) are a group of rare gentic disorders in which neuromuscular transmission is compromised by a variety of mechanisms, other than autoimmunity. Recently, substantial progress has been made by the identification of mutations in acetylcholine receptor (AChR) genes which cause CMS. We report on the clinical and genetic analysis of 18 independent CMS patients. All patients were clinically classified as sporadic cases of CMS (group III according to ENMC consensus). In order to investigate the prevalence of AChR mutations in this group we analyzed structural domains of the AChR genes at strategically important sites - the channel pore-lining regions (M2 domains) of the alpha, beta and epsilon subunits, and the extracellular domain close the acetylcholine (ACh) binding site. All patients showed wild-type sequence in these regions, mutations were not detected. Therefore, we conclude, that point mutations in domains which are known to cause slow channel congenital myasthenic syndromes (SCCMS) are rare in group III-patients in Germany. Determining the genetic defects causing CMS may have implications for diagnosis and genetic counseling of CMS patients. Moreover, this may be important for the therapeutic management of CMS as some patients may profit form quinidine sulfate. Therefore, further efforts will be undertaken to elucidate the underlying defects of CMS.     

Mutations in different functional domains of the human muscle acetylcholine receptor alpha subunit in patients with the slow-channel congenital myasthenic syndrome

Congenital myasthenic syndromes are a group of rare genetic disorders that compromise neuromuscular transmission. A subset of these disorders, the slow-channel congenital myasthenic syndrome (SCCMS), is dominantly inherited and has been shown to involve mutations within the muscle acetylcholine receptor (AChR). We have identified three new SCCMS mutations and a further familial case of the alpha G153S mutation. Single channel recordings from wild-type and mutant human AChR expressed in Xenopus oocytes demonstrate that each mutation prolongs channel activation episodes. The novel mutations alpha V156M, alpha T254I and alpha S269I are in different functional domains of the AChR alpha subunit. Whereas alpha T254I is in the pore-lining region, like five of six previously reported SCCMS mutations, alpha S269I and alpha V156M are in extracellular domains. alpha S269I lies within the short extracellular sequence between M2 and M3, and identifies a new region of muscle AChR involved in ACh binding/channel gating. alpha V156M, although located close to alpha G153S which has been shown to increase ACh binding affinity, appears to alter channel function through a different molecular mechanism. Our results demonstrate heterogeneity in the SCCMS, indicate new regions of the AChR involved in ACh binding/channel gating and highlight the potential role of mutations outside the pore-lining regions in altering channel function in other ion channel disorders.     

The non-peptide neuropeptide Y Y1 receptor antagonist BIBP3226 blocks the [Leu31,Pro34]neuropeptide Y-induced modulation of alpha 2-adrenoceptors in the nucleus tractus solitarii of the rat

The potential blockade of the neuropeptide Y (NPY) Y1 receptor agonist [Leu31,Pro34]NPY-induced modulation of the characteristics of alpha 2-adrenoceptor agonist [3H]p-aminoclonidine binding sites by a selective non-peptide NPY Y1 receptor antagonist BIBP3226, was studied in the nucleus tractus solitarii of the rat by means of quantitative receptor autoradiography. [Leu31,Pro34]NPY at a concentration of 10 nM significantly increased the Kd value of [3H]p-aminoclonidine binding sites in the nucleus tractus solitarii without influencing the Bmax, suggesting the existence of an antagonistic modulation by NPY Y1 receptors of alpha 2-adrenoceptors in the nucleus tractus solitarii. BIBP3226 at 100 nM fully blocked the [Leu31,Pro34]NPY-induced increase in Kd of the [3H]p-aminoclonidine binding sites. The present results therefore provide evidence, by use of a NPY Y1 receptor antagonist, for the existence of a NPY Y1/alpha 2 receptor interaction in the nucleus tractus solitarii.     

Characteristics of cyanopindolol analogues active at the beta 3-adrenoceptor in rat ileum

1. Cyanopindolol (CYP) is a potent antagonist at the beta 3-adrenoceptor in rat ileum. Several analogues of CYP and pindolol were synthesized that also produced antagonist effects at the beta 3-adrenoceptor. However, at high concentrations, these compounds appear to act as "partial agonists'. This study was conducted to determine the structural requirements of CYP analogues necessary for antagonist activity and to examine the possibility that the agonist effects of CYP and its analogues may occur through a mechanism independent of beta-adrenoceptor activation. 2. Analogues of CYP and pindolol were tested for antagonist activity in rat ileum in which the beta 1- and beta 2-adrenoceptors were blocked. Fourteen compounds were tested against (-)-isoprenaline, and four of the more potent analogues were then tested against BRL 37344. The two most potent antagonists were CYP and iodocyanopindolol. The pKb values (negative log of equilibrium dissociation constant) obtained against (-)-isoprenaline were significantly higher than those obtained against BRL 37344, but the cause of this difference is not known. 3. Several structural requirements were determined for antagonist activity. Modification at the carbon atom alpha to the secondary amine caused the antagonist potency to fall as the level of saturation was reduced. Thus, a quaternary carbon group, such as t-butyl, produced the most potent antagonist. Substitution with a large moiety such as a cyclohexyl or benzyl group reduced antagonist activity, probably due to steric hindrance. Inclusion of an electron-withdrawing group, such as a cyano or ethylester moiety, alpha to the indole nitrogen, also increased the potency. Iodination of CYP and ethylesterpindolol at the 3-position of the indole ring did not increase antagonist potency. In contrast, iodination of the almost inactive analogues produced a significant increase in potency, suggesting that a beneficial electronic effect on the indole ring imparted by the iodo moiety may be able to offset partially the negative effects caused by either the steric hindrance, of lack of a quaternary carbon alpha to the secondary amine. 4. Values for pseudo-pD2 were also determined by conducting cumulative concentration-response studies up to the limit of drug solubility. For nine of the compounds tested, the pKb was significantly higher than the pseudo-pD2 value. 5. The discrepancy between the pKb and pseudo-pD2 values was examined further. The agonist effects of iodocyanopindolol, the agonist with the highest potency, were not antagonized by CYP which was the most potent antagonist of (-)-isoprenaline and BRL 37344 at the beta 3-adrenoceptor. This suggests that the agonist effects of iodoCYP were produced through a different mechanism: either via another receptor, another isoform of the rat beta 3-adrenoceptor, or through a non-receptor-mediated effect. Pseudo-pD2 values did not correlate with log P values for these compounds, indicating that their relaxant effects were not simply a function of their lipid solubility. 6. This study has highlighted several structural requirements for antagonist binding potency at the rat ileum beta 3-adrenoceptor and should assist in the development of potent selective antagonists for this receptor.     

Synthesis of nitrodiazirinyl derivatives of neurotoxin II from Naja naja oxiana and their interaction with the Torpedo californica nicotinic acetylcholine receptor

Five singly modified nitrodiazirine derivatives of neurotoxin II (NT-II) from Naja naja oxiana were obtained after NT-II reaction with N-hydroxysuccinimide ester of (2-nitro-4-[3-(trifluoromethyl)-3H-diazirin-3yl]phenoxy)acet ic acid followed by chromatographic separation of the products. To localize the label positions, each derivative was first UV-irradiated and then subjected to reduction, carboxymethylation, and trypsinolysis. Tryptic digests were separated by reversed phase-HPLC, the labeled peptides being identified by mass spectrometry. The derivatives containing the photolabel at the position Lys 25, Lys 26, Lys 44, or Lys 46 were [125I]iodinated by the chloramine T procedure. Each iodinated derivative was found to form photoinduced cross-links with the membrane-bound nicotinic acetylcholine receptor (AChR) from Torpedo californica. The pattern of labeling the receptor's alpha, beta, gamma, or delta subunits was dependent on the photolabel position in the NT-II molecule and differed from that obtained earlier with an analogous series of p-azidobenzoyl derivatives of NT-II. The results obtained indicate that (i) different sides of the neurotoxin molecule are involved in the AChR binding, and (ii) fragments of the different AChR subunits are located close together at the neurotoxin-binding sites.     

Chronic GABA treatment downregulates the GABAA receptor alpha 2 and alpha 3 subunit mRNAS as well as polypeptide expression in primary cultured cerebral cortical neurons

Chronic GABA exposure of mammalian primary cultured cortical neurons results in a downregulation of the GABA-benzodiazepine receptor complex. In the present study, the mRNA levels, as well as polypeptide expression, for the GABAA receptor alpha 2 and alpha 3 subunits in cultured embryonic mouse cerebral cortical neurons (7 day old) were examined using northern analysis and immunoblotting techniques following chronic GABA treatment. The alpha 1 subunit mRNA or polypeptide could not be detected in these neurons. The steady state levels of mRNA for the GABAA receptor alpha 2 and alpha 3 subunits showed a decrease in comparison with untreated neurons. There was no change in the level of the beta actin or poly(A)+ RNA under the same experimental conditions. This agonist-induced reduction in the GABAA receptor alpha 2 and alpha 3 subunit mRNA was blocked by the concomitant exposure of neurons to R 5135, an antagonist of GABAA receptor. The polypeptide expression for the GABAA receptor alpha 2 and alpha 3 subunits in chronically GABA-treated neurons also showed a decline and this change was also blocked by the concomitant exposure of cells to GABA and R 5135. These results indicate that the chronic exposure of the GABAA receptor complex to agonist downregulates the expression of the alpha subunits of the receptor complex, which may be related to an observed decreases in the number of binding sites and GABA-induced 36Cl-influx in the cortical neurons.     

A review of mutagenesis studies of angiotensin II type 1 receptor, the three-dimensional receptor model in search of the agonist and antagonist binding site and the hypothesis of a receptor activation mechanism

OBJECTIVE: To seek the mechanism whereby agonists, competitive antagonists and insurmountable antagonists affect the receptor function differently, by reviewing recent mutagenesis studies of angiotensin II type 1 receptor (AT1) in which the binding of the agonist and antagonists and receptor signaling were affected. AT1 RECEPTOR STRUCTURE AND LIGAND BINDING SITES: We built a model of seven transmembrane spanning domains of the AT1 receptors using bacteriorhodopsin as a template. The carboxy terminal of angiotensin II binds to Lys199 in transmembrane domain 5, whereas the guanidinium group of Arg2 binds to Asp281 in transmembrane domain 7. Results of studies using mutagenesis supporting proposed ligand-docking models are discussed. HYPOTHESIS FOR THE LIGAND-INDUCED RECEPTOR SIGNALING MECHANISM: We submit a set of hypotheses for a mechanism whereby the ligand binding induces changes in the receptor conformation by the rotation of transmembrane helices as the initial event for the subsequent activation of a G protein. In this mechanism antagonists are not capable of rotating the helices but agonists are able to do so, which results in the formation of a hydrogen bond between Asp74 in transmembrane domain 2 and Tyr292 in transmembrane domain 7. This mechanism also provides plausible explanation for the activation of monoamine receptors. COMPETITIVE AND INSURMOUNTABLE ANTAGONISTS: Competitive antagonists share the same binding sites with agonists, but insurmountable antagonists do not, and binding of the latter does not preclude agonist binding, for example, to Asp281. CONCLUSION: This hypothesis of the intrareceptor signaling mechanism and the receptor model indicate that some amino acid residues essential for the signaling play their roles in the intrareceptor activation mechanism, whereas others participate directly in ligand binding.     

Distinctions in agonist and antagonist specificity conferred by anionic residues of the nicotinic acetylcholine receptor

Two anionic residues in the nicotinic acetylcholine receptor, Asp-152 in the alpha-subunit and Asp-174 in the gamma-subunit or the corresponding Asp-180 in the delta-subunit, are presumed to reside near the two agonist binding sites at the alphagamma and alphadelta subunit interfaces of the receptor and have been implicated in electrostatic attraction of cationic ligands. Through site-directed mutagenesis and analysis of state changes in the receptor elicited by agonists, we have distinguished the roles of anionic residues in conferring ligand specificity and ligand-induced state changes. alphaAsp-152 affects agonist and antagonist affinity similarly, whereas gammaAsp-174 and deltaAsp-180 primarily affect agonist affinity. Combining charge neutralization on the alpha subunit with that on the gamma and delta subunits shows an additivity in free energy changes for carbamylcholine and d-tubocurarine, suggesting independent contributions of these residues to stabilizing the bound ligands. Since both aromatic and anionic residues stabilize cationic ligands, we substituted tyrosines (Y) for the aspartyl residues. While the substitution, alphaD152Y, reduced the affinities for agonists and antagonists, the gammaD174Y/deltaD180Y mutations reduced the affinity for agonist binding, but surprisingly enhanced the affinity for d-tubocurarine. To ascertain whether selective changes in agonist binding stem from the capacity of agonists to form the desensitized state of the receptor, carbamylcholine binding was measured in the presence of an allosteric inhibitor, proadifen. Mutant nAChRs carrying alphaD152Q or gammaD174N/deltaD180N show similar reductions in dissociation constants for the desensitized compared with activable receptor state and a similar proadifen concentration dependence. Hence, these mutations influence ligand recognition rather than the capacity of the receptor to desensitize. By contrast, the alphaD200Q mutation diminishes the ratio of dissociation constants for two states and requires higher proadifen concentrations to induce desensitization. Thus, the contributions of alphaAsp-152, gamma/deltaAsp-174/180, and alphaAsp-200 in stabilizing ligand binding can be distinguished by the interactions between agonists and allosteric inhibitors.     

Alpha 2C-adrenoceptor-modulated release of noradrenaline in human right atrium

1. The aim of the present study was to characterize the presynaptic alpha 2-autoreceptors in human right atrium in terms of the alpha 2A-D system. Segments of atrial appendages were preincubated with [3H]-noradrenaline and then superfused in the presence of cocaine and stimulated electrically. pEC30% values of eight alpha-adrenoceptor antagonists with discriminatory power were determined. pEC30% is the negative logarithm of the antagonist concentration that increased the stimulation-induced overflow of tritium by 30%. For four antagonists, the dissociation constant KD was determined, in addition to pEC30%, against the overflow-inhibiting effect of 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) under autoinhibition-free conditions. 2. pEC30% and KD values yielded identical rank orders of antagonist affinity (rauwolscine > WB 4101 > phentolamine > prazosin) suggesting that both released noradrenaline and the exogenous agonist UK 14,304 activated the same receptor to inhibit release. 3. The eight antagonist pEC30% values obtained in right atrium correlated significantly with their pEC30% values, reported in the literature, at the presynaptic alpha 2C-autoreceptors in human kidney (r = 0.817; slope of the regression line 1.03). No significant correlation was obtained between pEC30% values at atrial autoreceptors and pKD values at previously characterized alpha 2A-autoreceptors in rabbit and alpha 2D-autoreceptors in rat, mouse and guinea-pig tissues. 4. Comparison of antagonist pEC30% values with their pKD values at native alpha 2 binding sites in cells or tissues that express a single subtype only, and with pKD values at alpha 2 binding sites in membranes of COS cells transfected with human alpha 2 subtype genes confirms the alpha 2C character of the atrial autoreceptors: significant correlations were obtained exclusively with the alpha 2C binding sites. 5. Ratios of KD values were computed for alpha 2-autoreceptors in human right atrium and for binding sites in COS cells transfected with human alpha 2 subtype genes. The autoreceptor ratios corresponded well with the respective ratios for the alpha 2C binding sites (maximal three fold deviation) but were, in part, markedly different from ratios calculated for alpha 2A and alpha 2B binding sites (up to 166 fold deviation). This outcome supports the alpha 2C designation of the autoreceptors. 6. In conclusion, the presynaptic alpha 2-autoreceptors in human right atrium are alpha 2C. In this they agree with the previously characterized alpha 2-autoreceptors in human kidney. The alpha 2C classification possibly separates, in general, human alpha 2-autoreceptors from those in lagomorph (rabbit) and rodent (rat, mouse, guinea pig) species that have been proposed to be predominantly alpha 2A or alpha 2D.