scholarly journals Tris+/Na+ permeability ratios of nicotinic acetylcholine receptors are reduced by mutations near the intracellular end of the M2 region.

1992 ◽  
Vol 99 (4) ◽  
pp. 545-572 ◽  
Author(s):  
B N Cohen ◽  
C Labarca ◽  
L Czyzyk ◽  
N Davidson ◽  
H A Lester
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Ion Selectivity ◽  
Point Mutations ◽  
Reversal Potential ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Mouse Muscle ◽  
Na Permeability ◽  
Delta Subunit

Tris+/Na+ permeability ratios were measured from shifts in the biionic reversal potentials of the macroscopic ACh-induced currents for 3 wild-type (WT), 1 hybrid, 2 subunit-deficient, and 25 mutant nicotinic receptors expressed in Xenopus oocytes. At two positions near the putative intracellular end of M2, 2' (alpha Thr244, beta Gly255, gamma Thr253, delta Ser258) and -1', point mutations reduced the relative Tris+ permeability of the mouse receptor as much as threefold. Comparable mutations at several other positions had no effects on relative Tris+ permeability. Mutations in delta had a greater effect on relative Tris+ permeability than did comparable mutations in gamma; omission of the mouse delta subunit (delta 0 receptor) or replacement of mouse delta with Xenopus delta dramatically reduced relative Tris+ permeability. The WT mouse muscle receptor (alpha beta gamma delta) had a higher relative permeability to Tris+ than the wild-type Torpedo receptor. Analysis of the data show that (a) changes in the Tris+/Na+ permeability ratio produced by mutations correlate better with the hydrophobicity of the amino acid residues in M2 than with their volume; and (b) the mole-fraction dependence of the reversal potential in mixed Na+/Tris+ solutions is approximately consistent with the Goldman-Hodgkin-Katz voltage equation. The results suggest that the main ion selectivity filter for large monovalent cations in the ACh receptor channel is the region delimited by positions -1' and 2' near the intracellular end of the M2 helix.

scholarly journals Roles of Amino Acids and Subunits in Determining the Inhibition of Nicotinic Acetylcholine Receptors by Competitive Antagonists

2007 ◽  
Vol 106 (6) ◽  
pp. 1186-1195 ◽  
Author(s):  
James P. Dilger ◽  
Ana Maria Vidal ◽  
Man Liu ◽  
Claire Mettewie ◽  
Takahiro Suzuki ◽  
...  
Keyword(s):  
Amino Acids ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Hek293 Cells ◽  
Wild Type ◽  
Binding Studies ◽  
Response Curves ◽  
Nicotinic Acetylcholine ◽  
Mouse Muscle ◽  
Epsilon Subunit

Background Binding sites for agonists and competitive antagonists (nondepolarizing neuromuscular blocking agents) are located at the alpha-delta and alpha-epsilon subunit interfaces of adult nicotinic acetylcholine receptors. Most information about the amino acids that participate in antagonist binding comes from binding studies with (+)-tubocurarine and metocurine. These bind selectively to the alpha-epsilon interface but are differentially sensitive to mutations. To test the generality of this observation, the authors measured current inhibition by five competitive antagonists on wild-type and mutant acetylcholine receptors. Methods HEK293 cells were transfected with wild-type or mutant (alphaY198F, epsilonD59A, epsilonD59N, epsilonD173A, epsilonD173N, deltaD180K) mouse muscle acetylcholine receptor complementary DNA. Outside-out patches were excised and perfused with acetylcholine in the absence and presence of antagonist. Concentration-response curves were constructed to determine antagonist IC50. An antagonist-removal protocol was used to determine dissociation and association rates. Results Effects of mutations were antagonist specific. alphaY198F decreased the IC50 of (+)-tubocurarine 10-fold, increased the IC50 of vecuronium 5-fold, and had smaller effects on other antagonists. (+)-Tubocurarine was the most sensitive antagonist to epsilonD173 mutations. epsilonD59 mutations had large effects on metocurine and cisatracurium. deltaD180K decreased inhibition by pancuronium, vecuronium, and cisatracurium. Inhibition by these antagonists was increased for receptors containing two delta subunits but no epsilon subunit. Differences in IC50 arose from differences in both dissociation and association rates. Conclusion Competitive antagonists exhibited different patterns of sensitivity to mutations. Except for pancuronium, the antagonists were sensitive to mutations at the alpha-epsilon interface. Pancuronium, vecuronium, and cisatracurium were selective for the alpha-delta interface. This suggests the possibility of synergistic inhibition by pairs of antagonists.

Mice lacking neuronal nicotinic acetylcholine receptor β4-subunit and mice lacking both α5- and β4-subunits are highly resistant to nicotine-induced seizures

2004 ◽  
Vol 17 (2) ◽  
pp. 221-229 ◽  
Author(s):  
Merav Kedmi ◽  
Arthur L. Beaudet ◽  
Avi Orr-Urtreger
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Mutant Mice ◽  
Wild Type ◽  
Nicotinic Acetylcholine ◽  
Nocturnal Frontal Lobe Epilepsy ◽  
Wide Range ◽  
High Doses ◽  
Dose Dependent ◽  
Rate Of Response

Nicotine, the main addictive component of tobacco, evokes a wide range of dose-dependent behaviors in rodents, and when administrated in high doses, it can induce clonic-tonic seizures. Nicotine acts through the nicotinic acetylcholine receptors (nAChRs). Mutations in the human α4- and the β2-nAChR subunit genes cause autosomal dominant nocturnal frontal lobe epilepsy. Using transgenic mice with mutations in nAChR subunits, it was demonstrated previously that the α4-, α5-, and α7-subunits are involved in nicotine-induced seizures. To examine the possibility that the β4-subunit is also involved in this phenotype, we tested mice with homozygous β4-subunit deficiency. The β4 null mice were remarkably resistant to nicotine-induced seizures compared with wild-type and α5 null mice. We also generated mice with double deficiency of both α5- and β4-nAChR subunits and demonstrated that they were more resistant to nicotine’s convulsant effect than either the α5 or the β4 single mutant mice. In addition, the single α5 mutants and the double α5β4-deficient mice exhibited a significantly shorter latency time to seizure than that of the wild-type mice. Our results thus show that β4-containing nAChRs have a crucial role in the pathogenesis of nicotine-induced seizures. Furthermore, by comparing multiple mutant mice with single and double subunit deficiency, we suggest that nicotinic receptors containing either α5- or β4-subunits are involved in nicotine-induced seizures and that receptors containing both subunits are likely to contribute to this phenomena as well. However, the α5-subunit, but not the β4-subunit, regulates the rate of response to high doses of nicotine.

scholarly journals Variants in CHRNB2 and CHRNA4 Identified in Patients with Insular Epilepsy

2020 ◽  
Vol 47 (6) ◽  
pp. 800-809 ◽  
Author(s):  
Maxime Cadieux-Dion ◽  
Simone Meneghini ◽  
Chiara Villa ◽  
Dènahin Hinnoutondji Toffa ◽  
Ronny Wickstrom ◽  
...  
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Functional Characterization ◽  
Reversal Potential ◽  
Missense Variant ◽  
Functional Expression ◽  
Functional Studies ◽  
Sorting Intolerant From Tolerant ◽  
First Case ◽  
And Function

Abstract:Purpose:Our purpose was to determine the role of CHRNA4 and CHRNB2 in insular epilepsy.Method:We identified two patients with drug-resistant predominantly sleep-related hypermotor seizures, one harboring a heterozygous missense variant (c.77C>T; p. Thr26Met) in the CHRNB2 gene and the other a heterozygous missense variant (c.1079G>A; p. Arg360Gln) in the CHRNA4 gene. The patients underwent electrophysiological and neuroimaging studies, and we performed functional characterization of the p. Thr26Met (c.77C>T) in the CHRNB2 gene.Results:We localized the epileptic foci to the left insula in the first case (now seizure-free following epilepsy surgery) and to both insulae in the second case. Based on tools predicting the possible impact of amino acid substitutions on the structure and function of proteins (sorting intolerant from tolerant and PolyPhen-2), variants identified in this report could be deleterious. Functional expression in human cell lines of α4β2 (wild-type), α4β2-Thr26Met (homozygote), and α4β2/β2-Thr26Met (heterozygote) nicotinic acetylcholine receptors revealed that the mutant subunit led to significantly higher whole-cell nicotinic currents. This feature was observed in both homo- and heterozygous conditions and was not accompanied by major alterations of the current reversal potential or the shape of the concentration-response relation.Conclusions:This study suggests that variants in CHRNB2 and CHRNA4, initially linked to autosomal dominant nocturnal frontal lobe epilepsy, are also found in patients with predominantly sleep-related insular epilepsy. Although the reported variants should be considered of unknown clinical significance for the moment, identification of additional similar cases and further functional studies could eventually strengthen this association.

scholarly journals Tetrapeptide Ac-HAEE-NH2 Protects α4β2 nAChR from Inhibition by Aβ

2020 ◽  
Vol 21 (17) ◽  
pp. 6272
Author(s):  
Evgeny P. Barykin ◽  
Aleksandra I. Garifulina ◽  
Anna P. Tolstova ◽  
Anastasia A. Anashkina ◽  
Alexei A. Adzhubei ◽  
...  
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Cholinergic Neurons ◽  
Xenopus Laevis Oocytes ◽  
Aβ Peptide ◽  
Amino Acid Residues ◽  
Cholinergic Dysfunction ◽  
Potential Binding ◽  
Electrode Voltage ◽  
Potential Binding Site

The cholinergic deficit in Alzheimer’s disease (AD) may arise from selective loss of cholinergic neurons caused by the binding of Aβ peptide to nicotinic acetylcholine receptors (nAChRs). Thus, compounds preventing such an interaction are needed to address the cholinergic dysfunction. Recent findings suggest that the 11EVHH14 site in Aβ peptide mediates its interaction with α4β2 nAChR. This site contains several charged amino acid residues, hence we hypothesized that the formation of Aβ-α4β2 nAChR complex is based on the interaction of 11EVHH14 with its charge-complementary counterpart in α4β2 nAChR. Indeed, we discovered a 35HAEE38 site in α4β2 nAChR, which is charge-complementary to 11EVHH14, and molecular modeling showed that a stable Aβ42-α4β2 nAChR complex could be formed via the 11EVHH14:35HAEE38 interface. Using surface plasmon resonance and bioinformatics approaches, we further showed that a corresponding tetrapeptide Ac-HAEE-NH2 can bind to Aβ via 11EVHH14 site. Finally, using two-electrode voltage clamp in Xenopus laevis oocytes, we showed that Ac-HAEE-NH2 tetrapeptide completely abolishes the Aβ42-induced inhibition of α4β2 nAChR. Thus, we suggest that 35HAEE38 is a potential binding site for Aβ on α4β2 nAChR and Ac-HAEE-NH2 tetrapeptide corresponding to this site is a potential therapeutic for the treatment of α4β2 nAChR-dependent cholinergic dysfunction in AD.

scholarly journals Functional Analysis of the Agrobacterium tumefaciens T-DNA Transport Pore Protein VirB8

2001 ◽  
Vol 183 (12) ◽  
pp. 3636-3641 ◽  
Author(s):  
Renu B. Kumar ◽  
Anath Das
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Point Mutations ◽  
Essential Amino Acids ◽  
Integral Membrane Protein ◽  
Tumor Formation ◽  
Dna Transfer ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Protein Protein Interaction ◽  
Pcr Method

ABSTRACT The VirB8 protein of Agrobacterium tumefaciens is essential for DNA transfer to plants. VirB8, a 237-residue polypeptide, is an integral membrane protein with a short N-terminal cytoplasmic domain. It interacts with two transport pore proteins, VirB9 and VirB10, in addition to itself. To study the role of these interactions in DNA transfer and to identify essential amino acids of VirB8, we introduced random mutations in virB8 by the mutagenic PCR method. The putative mutants were tested for VirB8 function by the ability to complement a virB8 deletion mutant in tumor formation assays. After multiple rounds of screening 13 mutants that failed to complement the virB8 deletion mutation were identified. Analysis of the mutant strains by DNA sequence analysis, Western blot assays, and reconstruction of new point mutations led to the identification of five amino acid residues that are essential for VirB8 function. The substitution of glycine-78 to serine, serine-87 to leucine, alanine-100 to valine, arginine-107 to proline or alanine, and threonine-192 to methionine led to the loss of VirB8 activity. When introduced into the wild-type strain, virB8 S87Lpartially suppressed the tumor forming ability of the wild-type protein. Analysis of protein-protein interaction by the yeast two-hybrid assay indicated that VirB8R107P is defective in interactions with both VirB9 and VirB10. A second mutant VirB8S87L is defective in interaction with VirB9.

scholarly journals Point Mutations in Exon I of the Herpes Simplex Virus Putative Terminase Subunit, UL15, Indicate that the Most Conserved Residues Are Essential for Cleavage and Packaging

2003 ◽  
Vol 77 (17) ◽  
pp. 9613-9621 ◽  
Author(s):  
Angela J. Przech ◽  
Dong Yu ◽  
Sandra K. Weller
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Point Mutations ◽  
Mutant Gene ◽  
Vero Cells ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Viral Genomes ◽  
Conserved Regions ◽  
Simplex Virus

ABSTRACT The herpes simplex virus UL15 and UL28 genes are believed to encode two subunits of the terminase involved in cleavage and packaging of viral genomes. Analysis of the UL15 protein sequence and its herpesvirus homologues revealed the presence of 20 conserved regions. Twelve of the twenty regions conserved among herpesviruses are also conserved in terminases from DNA bacteriophage. Point mutations in UL15 were designed in four conserved regions: L120N (CR1), Q205E (CR2), Q251E (CR3), G263A (CR3), and Y285S (CR4). Transfection experiments indicated that each mutant gene could produce stable UL15 protein at wild-type levels; however, only one mutant (Q251E) was able to complement the UL15-null virus. Each mutation was introduced into the viral genome by marker transfer, and all mutants except Q251E were unable to form plaques on Vero cells. Furthermore, failure to form plaques on Vero cells correlated with a defect in cleavage and packaging. Immunofluorescence experiments indicated that in cells infected with all mutant viruses the UL15 protein could be detected and was found to localize to replication compartments. Although wild-type and mutant Q251E were able to produce A, B, and C capsids, the rest of the mutants were only able to produce B capsids, a finding consistent with their defects in cleavage and packaging. In addition, all mutant UL15 proteins retained their ability to interact with B capsids. Therefore, amino acid residues 120, 205, 263, and 285 are essential for the cleavage and packaging process rather than for association with capsids or localization to replication compartments.

Actions of the insecticide 2 (nitromethylene) tetrahydro-1, 3-thiazine on insect and vertebrate nicotinic acetylcholine receptors

1989 ◽  
Vol 237 (1289) ◽  
pp. 501-514 ◽  
Keyword(s):  
Rat Brain ◽  
Glutamate Receptors ◽  
Acetylcholine Receptor ◽  
Nicotinic Acetylcholine Receptor ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Reversal Potential ◽  
Muscarinic Acetylcholine Receptors ◽  
Nicotinic Acetylcholine ◽  
Agonist Action

The nitromethylene heterocyclic compound 2(nitromethylene)tetrahydro) 1, 3-thiazine (NMTHT) inhibits the binding of [ 125 I) α -bungarotoxin to membranes prepared from cockroach ( Periplaneta americana ) nerve cord and fish ( Torpedo californica ) electric organ. Electrophysiological studies on the cockroach fast coxal depressor motorneuron (D f ) reveal a dose-dependent depolarization in response to bath-applied NMTHT. Responses to ionophoretic application of NMTHT on to the cell-body membrane of motorneuron D f are suppressed by bath-applied mecamylamine (1.0 x 10 -4 M) and α -bungarotoxin (1.0 x 10 -7 M). These findings, together with the detection of a reversal potential close to that estimated for acetylcholine, provide evidence for an agonist action of this nitromethylene on an insect neuronal nicotinic acetylcholine receptor. The binding of [ 3 H]H 12 -histrionicotoxin to Torpedo membranes was enhanced in the presence of NMTHT indicating an agonist action at this vertebrate peripheral nicotinic acetylcholine receptor. NMTHT is ineffective in radioligand binding assays for rat brain GABA A receptors, rat brain L-glutamate receptors and insect ( Musca domestica ) L-glutamate receptors. Partial block of rat brain muscarinic acetylcholine receptors is detected at millimolar concentrations of NMTHT. Thus nitromethylenes appear to exhibit selectivity for acetylcholine receptors and exhibitan agonist action at nicotinic acetylcholine receptors.

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