Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
J Gen Physiol
1998 Nov 01;1125:611-21. doi: 10.1085/jgp.112.5.611.
Show Gene links
Show Anatomy links
Delimiting the binding site for quaternary ammonium lidocaine derivatives in the acetylcholine receptor channel.
Pascual JM
,
Karlin A
.
???displayArticle.abstract???
The triethylammonium QX-314 and the trimethylammonium QX-222 are lidocaine derivatives that act as open-channel blockers of the acetylcholine (ACh) receptor. When bound, these blockers should occlude some of the residues lining the channel. Eight residues in the second membrane-spanning segment (M2) of the mouse-muscle alpha subunit were mutated one at a time to cysteine and expressed together with wild-type beta, gamma, and delta subunits in Xenopus oocytes. The rate constant for the reaction of each substituted cysteine with 2-aminoethyl methanethiosulfonate (MTSEA) was determined from the time course of the irreversible effect of MTSEA on the ACh-induced current. The reactions were carried out in the presence and absence of ACh and in the presence and absence of QX-314 and QX-222. These blockers had no effect on the reactions in the absence of ACh. In the presence of ACh, both blockers retarded the reaction of extracellularly applied MTSEA with cysteine substituted for residues from alphaVal255, one third of the distance in from the extracellular end of M2, to alphaGlu241, flanking the intracellular end of M2, but not with cysteine substituted for alphaLeu258 or alphaGlu262, at the extracellular end of M2. The reactions of MTSEA with cysteines substituted for alphaLeu258 and alphaGlu262 were considerably faster in the presence of ACh than in its absence. That QX-314 and QX-222 did not protect alphaL258C and alphaE262C against reaction with MTSEA in the presence of ACh implies that protection of the other residues was due to occlusion of the channel and not to the promotion of a less reactive state from a remote site. Given the 12-A overall length of the blockers and the alpha-helical conformation of M2 in the open state, the binding site for both blockers extends from alphaVal255 down to alphaSer248.
???displayArticle.pubmedLink???
9806969
???displayArticle.pmcLink???PMC2229442 ???displayArticle.link???J Gen Physiol ???displayArticle.grants???[+]
Figure 2. Aligned sequences of the M2 segments of mouse-muscle ACh receptor subunits. The numbering is that of the α subunit. The predicted membrane-spanning segments correspond to αM243 to αV261. The intracellular (IN) and extracellular (EX) ends are indicated. *Residues are aligned with residues in the ACh receptor from Torpedo electric tissue that were labeled by noncompetitive inhibitor derivatives. Mutations of the boxed-in residues affected channel block by QX-222. Cysteines substituted for the residues in bold italics are exposed in the channel in the presence of ACh (tested only in α and β). See text for references.
Figure 3. Structures of QX-314 and QX-222.
Figure 4. Inhibition of ACh-induced current in αL251C by QX-314 at different holding potentials. The top trace shows the holding potential. Both before and â¼3 s after the application of 25 μM ACh, the holding potential was stepped from â50 mV to â125, â75, â25, and back to â50 mV. The bottom trace shows the current. (inset) The peak ACh-induced current during the voltage steps minus the current during the voltage steps before ACh was added, with an expanded time axis. (A) No QX-314, (B) 0.1 mM QX-314, (C) 0.3 mM QX-314, (D) 1 mM QX-314. The four sections are part of a single experiment. Between the sections shown, the oocyte was washed and QX-314 at the indicated concentration was applied for 45 s before, and in addition to, the time indicated in the traces.
Figure 5. The effect of QX-314 on the irreversible inhibition of the ACh-induced current in two mutants. (A and B) αL258C. (C and D) αS248C. A and C show the test responses to ACh between applications of MTSEA in the presence of ACh and in the presence or absence of QX-314. B and D show the test currents as a function of cumulative exposure time to MTSEA. (âª) Initial responses and, in C and D, also extra final responses without any further application of MTSEA. (âµ) MTSEA in the presence of ACh. (â) MTSEA in the presence of both ACh and QX-314. ACh was applied at 25 μM to αL258C and at 100 μM to αS248C. 1 mM MTSEA was applied in 3-s pulses to αL258C, and 1.5 mM MTSEA was applied in 15-s pulses to αS248C. QX-314 was applied at 1 mM to αL258C and at 0.2 mM to αS248C. The calibration bars are 3 s and 5 μA.
Figure 6. The dependence on ACh of the protection by QX-314 against the irreversible inhibition by MTSEA. The mutant was αT244C. The log of the ACh-induced current is plotted against the cumulative time of application of MTSEA. (A) QX-314 added in the presence of ACh. (B) QX-314 added in the absence of ACh. (âª) Responses to 60 μM ACh before MTSEA was added. (â) Responses to ACh after 25-s applications of 2.5 μM MTSEA plus 60 μM ACh plus 0.1 mM QX-314. (âµ) Responses to ACh after 25-s applications of 2.5 μM MTSEA plus 60 μM ACh. (â´) Responses to ACh after 25-s applications of 250 μM MTSEA. (â¢) Responses to ACh after 25-s applications of 250 μM MTSEA plus 1 mM QX-314.
Figure 7. The dependence of protection on blocker concentration. The rate constant for the reaction of MTSEA in the presence of ACh at 10à EC50 and of the indicated concentration of QX-314 is divided by the rate constant in the presence of ACh but absence of QX-314. The mutants are αV255C (â¢) and αS248C (âª). The arrows mark the interpolated values of the relative rate constants at the IC50s for QX-314 inhibition of the ACh-induced current (Table I).
Figure 8. The protection by QX-314 and QX-222 of Cys-substituted residues in M2 against reaction with MTSEA. For each mutant, the rate constant of the reaction in the presence of channel blocker and ACh (kblocked) and the rate constant of the reaction in the presence of ACh alone (kopen) were determined as described in methods. The extent of protection is taken as 1 â (kblocked/kopen). The means of two to four determinations and average errors or SEM are plotted. The lighter bars represent the protection by QX-314, and the darker bars represent the protection by QX-222. *ND.
Figure 9. A model of QX-314 binding in the channel between two αM2 segments. The M2 segments are drawn as α helices. The dots represent the van der Waals surfaces. The drawing was made on a Silicon Graphics workstation running Insight II.
Adams,
Drug blockade of open end-plate channels.
1976, Pubmed
Adams,
Drug blockade of open end-plate channels.
1976,
Pubmed
Akabas,
Identification of acetylcholine receptor channel-lining residues in the M1 segment of the alpha-subunit.
1995,
Pubmed
,
Xenbase
Akabas,
Acetylcholine receptor channel structure probed in cysteine-substitution mutants.
1992,
Pubmed
,
Xenbase
Akabas,
Identification of acetylcholine receptor channel-lining residues in the entire M2 segment of the alpha subunit.
1994,
Pubmed
,
Xenbase
Anand,
Neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes have a pentameric quaternary structure.
1991,
Pubmed
,
Xenbase
Ascher,
The mode of action of antagonists of the excitatory response to acetylcholine in Aplysia neurones.
1978,
Pubmed
Boyd,
Desensitization of membrane-bound Torpedo acetylcholine receptor by amine noncompetitive antagonists and aliphatic alcohols: studies of [3H]acetylcholine binding and 22Na+ ion fluxes.
1984,
Pubmed
Charnet,
An open-channel blocker interacts with adjacent turns of alpha-helices in the nicotinic acetylcholine receptor.
1990,
Pubmed
Clapham,
Trifluoperazine reduces inward ionic currents and secretion by separate mechanisms in bovine chromaffin cells.
1984,
Pubmed
Colquhoun,
The modes of action of gallamine.
1981,
Pubmed
Cooper,
Pentameric structure and subunit stoichiometry of a neuronal nicotinic acetylcholine receptor.
1991,
Pubmed
DiPaola,
Mapping the alpha-subunit site photolabeled by the noncompetitive inhibitor [3H]quinacrine azide in the active state of the nicotinic acetylcholine receptor.
1990,
Pubmed
Farley,
Endplate channel block by guanidine derivatives.
1981,
Pubmed
Gage,
Some effects of procaine at the toad end-plate are not consistent with a simple channel-blocking model.
1984,
Pubmed
Gross,
Agitoxin footprinting the shaker potassium channel pore.
1996,
Pubmed
,
Xenbase
Heidmann,
Multiple sites of action for noncompetitive blockers on acetylcholine receptor rich membrane fragments from torpedo marmorata.
1983,
Pubmed
Herz,
Interaction of noncompetitive inhibitors with the acetylcholine receptor. The site specificity and spectroscopic properties of ethidium binding.
1987,
Pubmed
Hille,
Local anesthetics: hydrophilic and hydrophobic pathways for the drug-receptor reaction.
1977,
Pubmed
Horn,
Acetylcholine-induced current in perfused rat myoballs.
1980,
Pubmed
Horn,
Asymmetry of the acetylcholine channel revealed by quaternary anesthetics.
1980,
Pubmed
Hucho,
The ion channel of the nicotinic acetylcholine receptor is formed by the homologous helices M II of the receptor subunits.
1986,
Pubmed
Imoto,
Rings of negatively charged amino acids determine the acetylcholine receptor channel conductance.
1988,
Pubmed
,
Xenbase
Karlin,
Toward a structural basis for the function of nicotinic acetylcholine receptors and their cousins.
1995,
Pubmed
Karlin,
Explorations of the nicotinic acetylcholine receptor.
,
Pubmed
Karpen,
Cocaine and phencyclidine inhibition of the acetylcholine receptor: analysis of the mechanisms of action based on measurements of ion flux in the millisecond-to-minute time region.
1982,
Pubmed
Kirsch,
Differential effects of sulfhydryl reagents on saxitoxin and tetrodotoxin block of voltage-dependent Na channels.
1994,
Pubmed
Krodel,
Identification of a local anesthetic binding site in nicotinic post-synaptic membranes isolated from Torpedo marmorata electric tissue.
1979,
Pubmed
Leonard,
Evidence that the M2 membrane-spanning region lines the ion channel pore of the nicotinic receptor.
1988,
Pubmed
,
Xenbase
Lurtz,
Quinacrine and ethidium bromide bind the same locus on the nicotinic acetylcholine receptor from Torpedo californica.
1997,
Pubmed
Magleby,
A study of desensitization of acetylcholine receptors using nerve-released transmitter in the frog.
1981,
Pubmed
Maleque,
Meproadifen reaction with the ionic channel of the acetylcholine receptor: potentiation of agonist-induced desensitization at the frog neuromuscular junction.
1982,
Pubmed
Neher,
Local anaesthetics transiently block currents through single acetylcholine-receptor channels.
1978,
Pubmed
Neher,
The charge carried by single-channel currents of rat cultured muscle cells in the presence of local anaesthetics.
1983,
Pubmed
Oswald,
Multiple affinity states for noncompetitive blockers revealed by [3H]phencyclidine binding to acetylcholine receptor rich membrane fragments from Torpedo marmorata.
1983,
Pubmed
Pascual,
State-dependent accessibility and electrostatic potential in the channel of the acetylcholine receptor. Inferences from rates of reaction of thiosulfonates with substituted cysteines in the M2 segment of the alpha subunit.
1998,
Pubmed
,
Xenbase
Pascual,
Multiple residues specify external tetraethylammonium blockade in voltage-gated potassium channels.
1995,
Pubmed
,
Xenbase
Pedersen,
Structure of the noncompetitive antagonist-binding site of the Torpedo nicotinic acetylcholine receptor. [3H]meproadifen mustard reacts selectively with alpha-subunit Glu-262.
1992,
Pubmed
Ragsdale,
Molecular determinants of state-dependent block of Na+ channels by local anesthetics.
1994,
Pubmed
,
Xenbase
Revah,
The noncompetitive blocker [3H]chlorpromazine labels three amino acids of the acetylcholine receptor gamma subunit: implications for the alpha-helical organization of regions MII and for the structure of the ion channel.
1990,
Pubmed
Revah,
Mutations in the channel domain alter desensitization of a neuronal nicotinic receptor.
1991,
Pubmed
,
Xenbase
Ruff,
A quantitative analysis of local anaesthetic alteration of miniature end-plate currents and end-plate current fluctuations.
1977,
Pubmed
Sanchez,
Slow permeation of organic cations in acetylcholine receptor channels.
1986,
Pubmed
Schreibmayer,
Voltage clamping of Xenopus laevis oocytes utilizing agarose-cushion electrodes.
1994,
Pubmed
,
Xenbase
Sine,
Local anesthetics and histrionicotoxin are allosteric inhibitors of the acetylcholine receptor. Studies of clonal muscle cells.
1982,
Pubmed
Stauffer,
Electrostatic potential of the acetylcholine binding sites in the nicotinic receptor probed by reactions of binding-site cysteines with charged methanethiosulfonates.
1994,
Pubmed
Tamamizu,
Mutations in the M1 region of the nicotinic acetylcholine receptor alter the sensitivity to inhibition by quinacrine.
1995,
Pubmed
,
Xenbase
Wilson,
The location of the gate in the acetylcholine receptor channel.
1998,
Pubmed
Woodhull,
Ionic blockage of sodium channels in nerve.
1973,
Pubmed
Xu,
Interaction of picrotoxin with GABAA receptor channel-lining residues probed in cysteine mutants.
1995,
Pubmed
,
Xenbase
Zhang,
Identification of acetylcholine receptor channel-lining residues in the M1 segment of the beta-subunit.
1997,
Pubmed
,
Xenbase
Zhang,
Contribution of the beta subunit M2 segment to the ion-conducting pathway of the acetylcholine receptor.
1998,
Pubmed
,
Xenbase
