Mapping the alpha-subunit site photolabeled by the noncompetitive inhibitor [3H]quinacrine azide in the active state of the nicotinic acetylcholine receptor.

DiPaola, Mario; Kao, Peter N.; Karlin, Arthur

doi:10.1016/s0021-9258(19)38551-5

Cited by 92 publications

(20 citation statements)

References 74 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Most noncompetitive inhibitors exert little action unless the ion channel has been opened by an agonist (Adams, 1976;Changeux, 1990;Neely & Lingle, 1986;Neher & Steinbach, 1978;Ogden et al, 1981). Time-resolved photolabeling offered further evidence for the open state inhibition model with photoactive [3H]chlorpromazine and [3H]quinacrine azide as the inhibitor (Heidmann & Changeux, 1984; Heidmann & Changeux, 1986;DiPaola et al, 1990).…”

Section: Discussionmentioning

confidence: 99%

“…Under equilibrium conditions, noncompetitive inhibitors can label the nAcChoR in the resting state (in the absence of agonist) and the desensitized state (sustained application of agonist), but rapid labeling techniques must be used to label the transient open state before desensitization (Cox et al, 1984; Heidmann & Changeux, 1984;Muhn et al, 1984). Though the results obtained from these rapid labeling experiments were controversial because of the complication of receptor desensitization (Cox et al, 1985; Heidmann & Changeux, 1984Changeux, , 1986, there is strong evidence that the transient open state is labeled by [3 *H]chlorpromazine and [3H]quinacrine azide (DiPaola et al, 1990; Heidmann & Changeux, 1986). To date, photolabeling with [125I]TID has only been done under equilibrium conditions.…”

mentioning

confidence: 99%

See 1 more Smart Citation

A Hydrophobic Inhibitor of the Nicotinic Acetylcholine Receptor Acts on the Resting State

Raines²,

Miller³

1994

Biochemistry

View full text Add to dashboard Cite

3-(Trifluoromethyl)-3-(m-iodophenyl)diazirine (TID) has recently been found to be a noncompetitive inhibitor of the nicotinic acetylcholine receptor (nAcChoR) by both photolabeling and flux assays (White et al., 1991). However, these experiments were done when TID was in equilibrium with the nAcChoR, and thus only its interactions with the resting and the desensitized states of the nAcChoR were studied. In this work we characterized the interaction between TID and nAcChoR in the open and resting states using a flux assay. When TID and acetylcholine were simultaneously mixed with the nAcChoR in native Torpedo vesicles, TID did not inhibit agonist-induced 86Rb+ flux. However, following prolonged preincubation (4 min) of TID with nAcChoR, complete inhibition was observed with a half-inhibition constant of 0.4 microM TID and a Hill coefficient of 0.9. This suggested that TID might act either on the resting or the desensitized state in preference to the open state of the nAcChoR. Preincubation of nAcChoR with TID, followed by a 7 ms agonist-induced flux assay, showed that the flux response declined exponentially with preincubation time. Assuming a pseudo-first-order process, analysis revealed the rate constant for the onset of inhibition of nAcChoR in the resting state to be in the range of (1.2-3.4) x 10(6) M-1 s-1. To test if fast desensitization was enhanced by TID under these conditions, we used a fluorescent analog of acetylcholine. Stopped-flow fluorimetry showed that the fraction of nAcChoR in the predesensitized state did not increase during preincubation with TID.(ABSTRACT TRUNCATED AT 250 WORDS)

show abstract

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

A Hydrophobic Inhibitor of the Nicotinic Acetylcholine Receptor Acts on the Resting State

Raines²,

Miller³

1994

Biochemistry

View full text Add to dashboard Cite

show abstract

“…Not all evidence, however, points to the region between ␣ S248 and ␣ S252 as the sole binding site in the channel. Quinacrine azide photolabeled residues at the extracellular end of ␣ M1, specifically in the open state of the channel (Karlin, 1989;DiPaola et al, 1990), and mutations at the extracellular end of ␣ M1 affected quinacrine but not chlorpromazine binding (Tamamizu et al, 1995). Nevertheless, chlorpromazine and a number of other NCIs retarded the labeling by quina-crine azide (DiPaola et al, 1990) and competed with the binding of quinacrine (Lurtz et al, 1997).…”

Section: Introductionmentioning

confidence: 99%

Delimiting the Binding Site for Quaternary Ammonium Lidocaine Derivatives in the Acetylcholine Receptor Channel

Pascual

Karlin

1998

The Journal of General Physiology

View full text Add to dashboard Cite

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 α subunit were mutated one at a time to cysteine and expressed together with wild-type β, γ, and δ 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 αVal255, one third of the distance in from the extracellular end of M2, to αGlu241, flanking the intracellular end of M2, but not with cysteine substituted for αLeu258 or αGlu262, at the extracellular end of M2. The reactions of MTSEA with cysteines substituted for αLeu258 and αGlu262 were considerably faster in the presence of ACh than in its absence. That QX-314 and QX-222 did not protect αL258C and αE262C 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-Å overall length of the blockers and the α-helical conformation of M2 in the open state, the binding site for both blockers extends from αVal255 down to αSer248.

show abstract

“…(a) The noncompetitive antagonists [3H]chlorpromazine and [aH]triphenylmethylphosphonium photolabel residues in M2 (Giraudat, Dennis, Heidmann, Chang, and Changeux, 1986;Hucho, Oberthiir, and Lottspeich, 1986;Oberthtir, Muhn, Baumann, Lottspeich, Wittman-Liebold, and Hucho, 1986;Giraudat, Dennis, Heidmann, Haumont, Lederer, and Changeux, 1987;Giraudat, Galzi, Revah, Changeux, Haumont, and Lederer, 1989;Revah, Galzi, Giraudat, Haumont, Lederer, and Changeux, 1990); (b) mutations in M2 affect single-channel conductance and ion selectivity (Imoto, Methfessel, Sakmann, Mishina, Mori, Konno, Fukuda, Kurasaki, Bujo, Fujita, and Numa, 1986;Imoto, Busch, Sakmann, Mishina, Konno, Nakai, Bujo, Mori, Fukuda, and Numa, 1988;Leonard, Labarca, Charnet, Davidson, and Lester, 1988;Charnet, Labarca, Leonard, Vogelaar, Czyzyk, Gouin, Davidson, and Lester, 1990;Imoto, Konno, Nakai, Wang, Mishina, and Numa, 1991;Konno, Busch, von Kitzing, Imoto, Wang, Nakai, Mishina, Numa, and Sakmann, 1991;Villarroel, Herlitze, Koenen, and Sakmann, 1991;Cohen, Labarca, Czyzyk, Davidson, and Lester, 1992); and (c) mutations in M2 affect block of the nAChR by QX-222 (Leonard et al, 1988;Charnet et al, 1990). M1 may also form part of the pore lining (DiPaola, Kao, and Karlin, 1990).…”

Section: Introductionmentioning

confidence: 99%

Mutations in M2 alter the selectivity of the mouse nicotinic acetylcholine receptor for organic and alkali metal cations.

Cohen

Labarca

Davidson

et al. 1992

The Journal of General Physiology

116

View full text Add to dashboard Cite

We measured the permeability ratios (Px/PNa) of 3 wild-type, 1 hybrid, 2 subunit-deficient, and 22 mutant nicotinic receptors expressed in Xen0pus oocytes for alkali metal and organic cations using shifts in the biionic reversal potential of the macroscopic current. Mutations at three positions (2', 6', 10') in M2 affected ion selectivity. Mutations at position 2' (t~Thr244, 13Gly255, ~/Thr253, 8Ser258) near the intracellular end of M2 changed the organic cation permeability ratios as much as twofold and reduced Pc~/PNa and PK/PNa by 16-18%. Mutations at positions 6' and 10' increased the glycine ethyl ester/Na + and glycine methyl ester/Na + permeability ratios. Two subunit alterations also affected selectivity: omission of the 8 subunit reduced PcJPsa by 16%, and substitution ofXenopus 8 for mouse 8 increased P~nidi~ium/PNa more than twofold and reduced Pcs/PN~ by 34% and PLi/PN~ by 20%. The wild-type mouse receptor displayed a surprising interaction with the primary ammonium cations; relative permeability peaked at a chain length equal to four carbons. Analysis of the organic permeability ratios for the wild-type mouse receptor shows that (a) the diameter of the narrowest part of the pore is 8.4 ,~; (b) the mouse receptor departs significantly from size selectivity for monovalent organic cations; and (c) lowering the temperature reduces Pguanidinium/ PNa by 38% and Pbutylanunonitun/PNa more than twofold. The results reinforce present views that positions -1' and 2' are the narrowest part of the pore and suggest that positions 6' and 10' align some permeant organic cations in the pore in an interaction similar to that with channel blocker, QX-222.

show abstract

Mapping the alpha-subunit site photolabeled by the noncompetitive inhibitor [3H]quinacrine azide in the active state of the nicotinic acetylcholine receptor.

Cited by 92 publications

References 74 publications

A Hydrophobic Inhibitor of the Nicotinic Acetylcholine Receptor Acts on the Resting State

A Hydrophobic Inhibitor of the Nicotinic Acetylcholine Receptor Acts on the Resting State

Delimiting the Binding Site for Quaternary Ammonium Lidocaine Derivatives in the Acetylcholine Receptor Channel

Mutations in M2 alter the selectivity of the mouse nicotinic acetylcholine receptor for organic and alkali metal cations.

Contact Info

Product

Resources

About