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The extracellular linker of muscle acetylcholine receptor channels is a gating control element.

Grosman C, Salamone FN, Sine SM, Auerbach A - J. Gen. Physiol. (2000)

Bottom Line: With choline, this mutation accelerates channel opening approximately 28-fold, slows channel closing approximately 3-fold, but does not affect agonist binding to the closed state.Contrary to the alphaS269I mutation, Ile mutations at equivalent positions of the beta, straightepsilon, and delta subunits do not affect apparent open-channel lifetimes.The overall results indicate that this linker is a control element whose hydrophobicity determines channel gating in a position- and subunit-dependent manner.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, State University of New York at Buffalo, Buffalo, New York 14214, USA.

ABSTRACT
We describe the functional consequences of mutations in the linker between the second and third transmembrane segments (M2-M3L) of muscle acetylcholine receptors at the single-channel level. Hydrophobic mutations (Ile, Cys, and Phe) placed near the middle of the linker of the alpha subunit (alphaS269) prolong apparent openings elicited by low concentrations of acetylcholine (ACh), whereas hydrophilic mutations (Asp, Lys, and Gln) are without effect. Because the gating kinetics of the alphaS269I receptor (a congenital myasthenic syndrome mutant) in the presence of ACh are too fast, choline was used as the agonist. This revealed an approximately 92-fold increased gating equilibrium constant, which is consistent with an approximately 10-fold decreased EC(50) in the presence of ACh. With choline, this mutation accelerates channel opening approximately 28-fold, slows channel closing approximately 3-fold, but does not affect agonist binding to the closed state. These ratios suggest that, with ACh, alphaS269I acetylcholine receptors open at a rate of approximately 1.4 x 10(6) s(-1) and close at a rate of approximately 760 s(-1). These gating rate constants, together with the measured duration of apparent openings at low ACh concentrations, further suggest that ACh dissociates from the diliganded open receptor at a rate of approximately 140 s(-1). Ile mutations at positions flanking alphaS269 impair, rather than enhance, channel gating. Inserting or deleting one residue from this linker in the alpha subunit increased and decreased, respectively, the apparent open time approximately twofold. Contrary to the alphaS269I mutation, Ile mutations at equivalent positions of the beta, straightepsilon, and delta subunits do not affect apparent open-channel lifetimes. However, in beta and straightepsilon, shifting the mutation one residue to the NH(2)-terminal end enhances channel gating. The overall results indicate that this linker is a control element whose hydrophobicity determines channel gating in a position- and subunit-dependent manner. Characterization of the transition state of the gating reaction suggests that during channel opening the M2-M3L of the alpha subunit moves before the corresponding linkers of the beta and straightepsilon subunits.

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Mentions: Where k−/k+ (= Kd) and j−/j+ (= Jd) are the microscopic agonist-dissociation equilibrium constants from the closed and open channels, respectively, β0/α0 (= θ0), β1/α1 (= θ1), and β2/α2 (= θ2) are the gating equilibrium constants of un-, mono-, and diliganded receptors, respectively, k+D/k−D is the desensitization equilibrium constant, and k+g/k−g is the equilibrium constant between OA2 and CA2*. Because this model was used to interpret data recorded at low concentrations of ACh, blockade by the agonist itself was ignored. According to Fig. 2, to satisfy detailed balance, θ1 = θ0Kd/Jd and θ2 = θ0(Kd/Jd)2.


The extracellular linker of muscle acetylcholine receptor channels is a gating control element.

Grosman C, Salamone FN, Sine SM, Auerbach A - J. Gen. Physiol. (2000)

© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2233691&req=5

Mentions: Where k−/k+ (= Kd) and j−/j+ (= Jd) are the microscopic agonist-dissociation equilibrium constants from the closed and open channels, respectively, β0/α0 (= θ0), β1/α1 (= θ1), and β2/α2 (= θ2) are the gating equilibrium constants of un-, mono-, and diliganded receptors, respectively, k+D/k−D is the desensitization equilibrium constant, and k+g/k−g is the equilibrium constant between OA2 and CA2*. Because this model was used to interpret data recorded at low concentrations of ACh, blockade by the agonist itself was ignored. According to Fig. 2, to satisfy detailed balance, θ1 = θ0Kd/Jd and θ2 = θ0(Kd/Jd)2.

Bottom Line: With choline, this mutation accelerates channel opening approximately 28-fold, slows channel closing approximately 3-fold, but does not affect agonist binding to the closed state.Contrary to the alphaS269I mutation, Ile mutations at equivalent positions of the beta, straightepsilon, and delta subunits do not affect apparent open-channel lifetimes.The overall results indicate that this linker is a control element whose hydrophobicity determines channel gating in a position- and subunit-dependent manner.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, State University of New York at Buffalo, Buffalo, New York 14214, USA.

ABSTRACT
We describe the functional consequences of mutations in the linker between the second and third transmembrane segments (M2-M3L) of muscle acetylcholine receptors at the single-channel level. Hydrophobic mutations (Ile, Cys, and Phe) placed near the middle of the linker of the alpha subunit (alphaS269) prolong apparent openings elicited by low concentrations of acetylcholine (ACh), whereas hydrophilic mutations (Asp, Lys, and Gln) are without effect. Because the gating kinetics of the alphaS269I receptor (a congenital myasthenic syndrome mutant) in the presence of ACh are too fast, choline was used as the agonist. This revealed an approximately 92-fold increased gating equilibrium constant, which is consistent with an approximately 10-fold decreased EC(50) in the presence of ACh. With choline, this mutation accelerates channel opening approximately 28-fold, slows channel closing approximately 3-fold, but does not affect agonist binding to the closed state. These ratios suggest that, with ACh, alphaS269I acetylcholine receptors open at a rate of approximately 1.4 x 10(6) s(-1) and close at a rate of approximately 760 s(-1). These gating rate constants, together with the measured duration of apparent openings at low ACh concentrations, further suggest that ACh dissociates from the diliganded open receptor at a rate of approximately 140 s(-1). Ile mutations at positions flanking alphaS269 impair, rather than enhance, channel gating. Inserting or deleting one residue from this linker in the alpha subunit increased and decreased, respectively, the apparent open time approximately twofold. Contrary to the alphaS269I mutation, Ile mutations at equivalent positions of the beta, straightepsilon, and delta subunits do not affect apparent open-channel lifetimes. However, in beta and straightepsilon, shifting the mutation one residue to the NH(2)-terminal end enhances channel gating. The overall results indicate that this linker is a control element whose hydrophobicity determines channel gating in a position- and subunit-dependent manner. Characterization of the transition state of the gating reaction suggests that during channel opening the M2-M3L of the alpha subunit moves before the corresponding linkers of the beta and straightepsilon subunits.

Show MeSH
Related in: MedlinePlus