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.

Biophysical and pharmacological characterization of α6-containing nicotinic acetylcholine receptors expressed in HEK293 cells

2014 ◽  
Vol 1542 ◽  
pp. 1-11 ◽  
Author(s):  
Andreas H. Rasmussen ◽  
Dorte Strøbæk ◽  
Tino Dyhring ◽  
Marianne L. Jensen ◽  
Dan Peters ◽  
...  
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Hek293 Cells ◽  
Pharmacological Characterization ◽  
Nicotinic Acetylcholine

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.

Differential Sensitivities of Mammalian Neuronal and Muscle Nicotinic Acetylcholine Receptors to General Anesthetics 

1997 ◽  
Vol 86 (4) ◽  
pp. 866-874 ◽  
Author(s):  
J. M. Violet ◽  
D. L. Downie ◽  
R. C. Nakisa ◽  
W. R. Lieb ◽  
N. P. Franks
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Xenopus Oocytes ◽  
General Anesthetics ◽  
Response Curves ◽  
Nicotinic Acetylcholine ◽  
Considerable Uncertainty ◽  
Messenger Ribonucleic Acid ◽  
The Central Nervous System ◽  
Type 3

Background Nicotinic acetylcholine receptors (nAChRs) are members of a superfamily of fast neurotransmitter-gated receptor channels that includes the gamma-aminobutyric acidA (GABAA), glycine and serotonin type 3 (5-HT3) receptors. Most previous work on the interactions of general anesthetics with nAChRs has involved the muscle-type receptor. The authors investigate the effects of general anesthetics on defined mammalian neuronal and muscle nAChRs expressed in Xenopus oocytes. Methods Complementary deoxyribonucleic acid (cDNA) or messenger ribonucleic acid (mRNA) encoding for various neuronal or muscle nAChR subunits was injected into Xenopus oocytes, and the resulting ACh-activated currents were studied using the two-electrode voltage-clamp technique. The effects of halothane, isoflurane, sevoflurane, and propofol on the peak acetylcholine-induced currents were investigated, and concentration-response curves were constructed. Results The neuronal nAChRs were found to be much more sensitive to general anesthetics than were the muscle nAChRs, with IC50 concentrations being 10- to 35-fold less for the neuronal receptors. For the inhalational general anesthetics, the IC50 concentrations were considerably less than the free aqueous concentrations that cause general anesthesia in mammals. In addition, qualitative (dependence on acetylcholine concentration) and quantitative (steepness of concentration-response curves) differences in the anesthetic interactions between the neuronal and muscle nAChRs suggest that different mechanisms of inhibition may be involved. Conclusions Although there is considerable uncertainty about the physiologic roles that neuronal nAChRs play in the central nervous system, their extraordinary sensitivity to general anesthetics, particularly the inhalational agents, suggests they may mediate some of the effects of general anesthetics at surgical, or even subanesthetic, concentrations.

Sign in / Sign up

Export Citation Format

Share Document