Genetic disorders caused by mutated acetylcholine receptors

The nicotinic acetylcholine receptors (nAChRs) are members of the large family of ligand-gated ion channels and are constituted by the assembly of five subunits arranged pseudosymmetrically around the central axis that forms a cation-selective ion pore. They are widely distributed in both the nervous system and non-neuronal tissues, and can be activated by endogenous agonists such as acetylcholine or exogenous ligands such as nicotine. Mutations in neuronal nAChRs are found in a rare form of familial nocturnal frontal lobe epilepsy (ADNFLE), while mutations in the neuromuscular subtype of the nAChR are responsible for either congenital myasthenia syndromes (adult subtype of neuromuscular nAChR) or a form of arthrogryposis multiplex congenita type Escobar (fetal subtype of neuromuscular nAChR).     

N-substituted derivatives of 4-piperidinyl benzilate: affinities for brain muscarinic acetylcholine receptors

N-Substituted derivatives of 4-piperidinyl benzilate were synthesized and their affinities for central muscarinic cholinergic receptors determined using an in vitro radioligand binding assay. 4-Piperidinyl benzilate exhibited a Ki value of 2.0 nM. N-Substitution with a methyl or an ethyl group increased the affinity to 0.2 nM, whereas substitution with a n-propyl or isopropyl group decreased the binding affinity over 100 fold. Compounds with aralkyl substitutions at the nitrogen atom of piperidinyl benzilate were also synthesized and evaluated. The Ki values (nM) obtained for these compounds were: benzyl, 0.2; p-nitrobenzyl, 13.0; p-fluorobenzyl, 3.0; phenethyl, 8.0; p-nitrophenethyl, 15.0. These data suggest that a binding region near the piperidinyl nitrogen may tolerate bulky aromatic substitutions (e.g., benzyl or phenethyl) as well or better than straight chain or branched alkyl substitutions (e.g., n-propyl or isopropyl).     

Long release latencies are increased by acetylcholine at frog endplate

Uni-quantal endplate currents (EPCs) were recorded extracellularly at the frog neuromuscular synapse and their latency dispersions expressed as P(90) were estimated in the presence of acetylcholine. Stimulation-evoked EPCs with long release latencies increased in number when acetylcholine was applied. P90, which is designated as the interval between the minimal synaptic delay and the time at which 90 per cent of all measured uni-quantal EPCs had occurred, was significantly and reversibly increased by 66 per cent from 0.51 ms to 0.85 ms in the presence of 5x10(-4) M acetylcholine. This indicates that the evoked release pattern is less synchronous and the increased asynchrony leads to a substantial drop (by 28 per cent) in the amplitude of reconstructed multi-quantal currents.     

Permeability of the endplate membrane activated by acetylcholine to some organic cations

The ability of various organic cations to depolarize the ACh-activated endplate membrane in the absence of Na ions was examined on frog sartorius muscle by measuring the endplate potential on the muscle surface with the moving electrode technique. The ACh-activated endplate membrane was very permeable to ammonium and its methyl and hydroxy derivatives, and moderately permeable to guanidine derivatives and Tris (hydroxymethyl) aminomethane. The permeability of alkylol derivatives of ammonium diminished progressively with increase in molecular size. The present results suggested that the endplate ionic channels can be represented by a pore of about 6.4 Å in diameter.     

Permeability of the endplate membrane activated by acetylcholine to some organic cations.

The ability of various organic cations to depolarize the ACh-activated endplate membrane in the absence of Na ions was examined on frog sartorius muscle by measuring the endplate potential on the muscle surface with the moving electrode technique. The ACh-activated endplate membrane was very permeable to ammonium and its methyl and hydroxy derivatives, and moderately permeable to guanidine derivatives and Tris (hydroxymethyl) aminomethane. The permeability of alkylol derivatives of ammonium diminished progressively with increase in molecular size. The present results suggested that the endplate ionic channels can be represented by a pore of about 6.4 A in diameter.     

A motif present in the main cytoplasmic loop of nicotinic acetylcholine receptors and catalases

A motif containing five conserved amino acids (RXPXTH(X)14P) was detected in 111 proteins, including 82 nicotinic acetylcholine receptor (nAChR) subunits and 20 catalases. To explore possible functional roles of this motif in nAChRs two approaches were used: first, the motif sequences in nAChR subunits and catalases were analysed and compared; and, second, deletions in the rat alpha2 and beta4 nAChR subunits expressed in Xenopus oocytes were analysed. Compared to the three-dimensional structure of bovine hepatic catalase, structural coincidences were found in the motif of catalases and nAChRs. On the other hand, partial deletions of the motif in the alpha2 or beta4 subunits and injection of the mutants into oocytes was followed by a very weak expression of functional nAChRs; oocytes injected with alpha2 and beta4 subunits in which the entire motif had been deleted failed to elicit any acetylcholine currents. The results suggest that the motif may play a role in the activation of nAChRs.     

Reversal of agonist selectivity by mutations of conserved amino acids in the binding site of nicotinic acetylcholine receptors

Homomeric alpha7 and heteromeric alpha4beta2 nicotinic acetylcholine receptors (nAChR) can be distinguished by their pharmacological properties, including agonist specificity. We introduced point mutations of conserved amino acids within the C loop, a region of the receptor critical for agonist binding, and we examined the expression of the mutant receptors in Xenopus oocytes. Mutation of either a conserved C loop tyrosine (188) to phenylalanine or a nearby conserved aspartate (197) to alanine resulted in alpha7 receptors for which the alpha7-selective agonist 3-(4-hydroxy, 2-methoxybenzylidene) anabaseine (4OH-GTS-21) had roughly the same potency as for wild-type receptors, whereas the physiologic agonist acetylcholine (ACh) showed drastically reduced potency for these mutant receptors. Corresponding mutations in alpha4 receptors co-expressed with beta2 resulted in alpha4beta2 receptors for which ACh potency was relatively unchanged, although the efficacy of the alpha7-selective agonist 4OH-GTS-21 was increased greatly relative to that of ACh. We also investigated the significance of a conserved lysine (145 in alpha7), proposed to form a stable salt bridge with Asp-197 in the resting state of the receptor. Mutations of this residue in both alpha7 and alpha4 resulted in receptors that were largely unresponsive to both ACh and 4OH-GTS-21. Our results suggest that initiation of gating depends both on specific interactions between residues in the C loop domain and, depending on receptor subtype, the physiochemical properties of the agonist, so that in the altered environment of the alpha4Y190F-binding site, large hydrophobic benzylidene anabaseines may close the C loop and initiate channel gating more effectively than the polar agonist ACh.     

Immobilization of nicotinic acetylcholine receptors in mouse C2 myotubes by agrin-induced protein tyrosine phosphorylation

Agrin induces the formation of highly localized specializations on myotubes at which nicotinic acetylcholine receptors (AChRs) and many other components of the postsynaptic apparatus at the vertebrate skeletal neuromuscular junction accumulate. Agrin also induces AChR tyrosine phosphorylation. Treatments that inhibit tyrosine phosphorylation prevent AChR aggregation. To examine further the relationship between tyrosine phosphorylation and receptor aggregation, we have used the technique of fluorescence recovery after photobleaching to assess the lateral mobility of AChRs and other surface proteins in mouse C2 myotubes treated with agrin or with pervanadate, a protein tyrosine phosphatase inhibitor. Agrin induced the formation of patches in C2 myotubes that stained intensely with anti-phosphotyrosine antibodies and within which AChRs were relatively immobile. Pervanadate, on the other hand, increased protein tyrosine phosphorylation throughout the myotube and caused a reduction in the mobility of diffusely distributed AChRs, without affecting the mobility of other membrane proteins. Pervanadate, like agrin, caused an increase in AChR tyrosine phosphorylation and a decrease in the rate at which AChRs could be extracted from intact myotubes by mild detergent treatment, suggesting that immobilized receptors were phosphorylated and therefore less extractable. Indeed, phosphorylated receptors were extracted from agrin-treated myotubes more slowly than nonphosphorylated receptors. AChR aggregates at developing neuromuscular junctions in embryonic rat muscles also labeled with anti- phosphotyrosine antibodies, suggesting that tyrosine phosphorylation could mediate AChR aggregation in vivo as well. Thus, agrin appears to induce AChR aggregation by creating circumscribed domains of increased protein tyrosine phosphorylation within which receptors become phosphorylated and immobilized.     

Regulation of nicotinic acetylcholine receptors by protein phosphorylation

Neurotransmitter receptors and ion channels play a critical role in the transduction of signals at chemical synapses. The modulation of neurotransmitter receptor and ion channel function by protein phosphorylation is one of the major regulatory mechanisms in the control of synaptic transmission. The nicotinic acetylcholine receptor (nAcChR) has provided an excellent model system in which to study the modulation of neurotransmitter receptors and ion channels by protein phosphorylation since the structure and function of this receptor have been so extensively characterized. In this article, the structure of the nAcChR from the electric organ of electric fish, skeletal muscle, and the central and peripheral nervous system will be briefly reviewed. Emphasis will be placed on the regulation of the phosphorylation of nAcChR by second messengers and by neurotransmitters and hormones. In addition, recent studies on the functional modulation of nicotinic receptors by protein phosphorylation will be reviewed.     

Alkaptonuria caused by compound heterozygote mutations

Alkaptonuria is a rare autosomal recessive disorder of inborn errors of metabolism. It is characterised by the deposition of "ochronotic pigment" especially in connective tissue as a result of deficieny of the "homogentisic acid oxidase" enzyme which has a role in the catabolism of tyrosine and phenylalanine. A compound heterozygote alkaptonuria patient, with manifestations in adulthood, without infantile and childhood signs is presented. The described alkaptonuria mutations are reported for the first time in the Turkish population.     

Activation of solubilized G-proteins by muscarinic acetylcholine receptors

Binding of GTP and its analogue, guanosine 5′-O-[γ-thio]triphosphate (GTP[S]) to G-proteins, and release of GTP[S] from G-proteins are stimulated by muscarinic acetylcholine (mACh) receptors in intact cardiac membranes. Upon solubilization of receptors and G-proteins by membrane extraction with the detergent, 3-[(cholamidopropyl)dimethylammonio]-1-propanesulphonate, followed by sucrose density gradient centrifugation, agonist-liganded mACh receptors stimulated binding of GTP[S] and hydrolysis of GTP by G-proteins with similar requirements as in intact membranes. One soluble agonist-activated mACh receptor induced binding of GTP[S] to several (about seven) soluble G-proteins. In contrast to intact membranes, however, agonist activation of mACh receptors did not induce release of GTP[S] from solubilized G-proteins. The data presented indicate that mACh receptors can interact with and efficiently activate G-proteins even in solution, whereas the possible interaction of receptors with GTP[S]-liganded G-proteins observed in intact membranes is lost upon solubilization of these components.     

Purification of muscarinic acetylcholine receptors by affinity chromatography

Calf forebrain homogenates contain 2.8 pM muscarinic acetylcholine receptors per mg of protein. [3H]Antagonist saturation binding experiments under equilibrium conditions revealed a single class of sites with equilibrium dissociation constants of 0.82 nM for [3H]dexetimide and 0.095 nM for [3H]quinuclidinyl benzilate. Displacement binding studies with agonists revealed the presence of low and high affinity sites. Here we describe the solubilization of muscarinic acetylcholine receptors with digitonin and their purification by affinity chromatography using an affinity gel which consisted of dexetimide coupled to Affi-Gel 10 (i.e., carboxy N-hydroxysuccinimide esters linked via a 1 nm spacer arm to agarose beads). Purified proteins were obtained by specific elution with muscarinic drugs, i.e., the antagonist atropine and the irreversible ligand propylbenzilylcholine mustard. SDS-polyacrylamide gel electrophoresis of the radioiodinated purified preparations revealed a major 70-K protein.     

Impairment of protein degradation in myofibrillar myopathy caused by FLNC/filamin C mutations

Myofibrillar myopathy caused by FLNC/filamin C mutations is characterized by disintegration of myofibrils and a massive formation of protein aggregates within skeletal muscle fibers. We performed immunofluorescence studies in skeletal muscle sections from filaminopathy patients to detect disturbances of protein quality control mechanisms. Our analyses revealed altered expression of chaperone proteins and components of proteasomal and autophagic degradation pathways in abnormal muscle fibers that harbor protein deposits but not in neighboring muscle fibers without pathological protein aggregation. These findings suggest a dysfunction of protein stabilizing and degrading mechanisms that leads to a pathological accumulation of protein aggregates in abnormal fibers. Accordingly, a pharmacological modulation of chaperone activity may be a promising therapeutic strategy to prevent protein aggregation and to reduce disease progression. Newly established filaminopathy cell culture models provide a suitable basis for testing such pharmacological approaches.     

Compensatory mutations restore the replication defects caused by cytotoxic T lymphocyte escape mutations in hepatitis C virus polymerase

While human leukocyte antigen B57 (HLA-B57) is associated with the spontaneous clearance of hepatitis C virus (HCV), the mechanisms behind this control remain unclear. Immunodominant CD8(+) T cell responses against the B57-restricted epitopes comprised of residues 2629 to 2637 of nonstructural protein 5B (NS5B(2629-2637)) (KSKKTPMGF) and E2(541-549) (NTRPPLGNW) were recently shown to be crucial in the control of HCV infection. Here, we investigated whether the selection of deleterious cytotoxic T lymphocyte (CTL) escape mutations in the NS5B KSKKTPMGF epitope might impair viral replication and contribute to the B57-mediated control of HCV. Common CTL escape mutations in this epitope were identified from a cohort of 374 HCV genotype 1a-infected subjects, and their impact on HCV replication assessed using a transient HCV replicon system. We demonstrate that while escape mutations at residue 2633 (position 5) of the epitope had little or no impact on HCV replication in vitro, mutations at residue 2629 (position 1) substantially impaired replication. Notably, the deleterious mutations at position 2629 were tightly linked in vivo to upstream mutations at residue 2626, which functioned to restore the replicative defects imparted by the deleterious escape mutations. These data suggest that the selection of costly escape mutations within the immunodominant NS5B KSKKTPMGF epitope may contribute in part to the control of HCV replication in B57-positive individuals and that persistence of HCV in B57-positive individuals may involve the development of specific secondary compensatory mutations. These findings are reminiscent of the selection of deleterious CTL escape and compensatory mutations by HLA-B57 in HIV-1 infection and, thus, may suggest a common mechanism by which alleles like HLA-B57 mediate protection against these highly variable pathogens.     

Meconium ileus caused by mutations in GUCY2C, encoding the CFTR-activating guanylate cyclase 2C

Meconium ileus, intestinal obstruction in the newborn, is caused in most cases by CFTR mutations modulated by yet-unidentified modifier genes. We now show that in two unrelated consanguineous Bedouin kindreds, an autosomal-recessive phenotype of meconium ileus that is not associated with cystic fibrosis (CF) is caused by different homozygous mutations in GUCY2C, leading to a dramatic reduction or fully abrogating the enzymatic activity of the encoded guanlyl cyclase 2C. GUCY2C is a transmembrane receptor whose extracellular domain is activated by either the endogenous ligands, guanylin and related peptide uroguanylin, or by an external ligand, Escherichia coli (E. coli) heat-stable enterotoxin STa. GUCY2C is expressed in the human intestine, and the encoded protein activates the CFTR protein through local generation of cGMP. Thus, GUCY2C is a likely candidate modifier of the meconium ileus phenotype in CF. Because GUCY2C heterozygous and homozygous mutant mice are resistant to E. coli STa enterotoxin-induced diarrhea, it is plausible that GUCY2C mutations in the desert-dwelling Bedouin kindred are of selective advantage.