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 α subunit

Juan M. Pascual, Arthur Karlin

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101 Citations (Scopus)

Abstract

Ion channel function depends on the chemical and physical properties and spatial arrangement of the residues that line the channel lumen and on the electrostatic potential within the lumen. We have used small, sulfhydryl- specific thiosulfonate reagents, both positively charged and neutral, to probe the environment within the acetylcholine (ACh) receptor channel. Rate constants were determined for their reactions with cysteines substituted for nine exposed residues in the second membrane-spanning segment (M2) of the α subunit. The largest rate constants, both in the presence and absence of ACh, were for the reactions with the cysteine substituted for αThr244, near the intracellular end of the channel. In the open state of the channel, but not in the closed state, the rate constants for the reactions of the charged reagents with several substituted cysteines depended on the transmembrane electrostatic potential, and the electrical distance of these cysteines increased from the extracellular to the intracellular end of M2. Even at zero transmembrane potential, the ratios of the rate constants for the reactions of three positively charged reagents with αT244C, αL251C, and αL258C to the rate constant for the reaction of an uncharged reagent were much greater in the open than in the closed state. This dependence of the rate constants on reagent charge is consistent with an intrinsic electrostatic potential in the channel that is considerably more negative in the open state than in the closed state. The effects of ACh on the rate constants for the reactions of substituted Cys along the length of αM2, on the dependence of the rate constants on the transmembrane potential, and on the intrinsic potential support a location of a gate more intracellular than αThr244.

Original languageEnglish (US)
Pages (from-to)717-739
Number of pages23
JournalJournal of General Physiology
Volume111
Issue number6
DOIs
StatePublished - Jun 1998

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Cholinergic Receptors
Static Electricity
Cysteine
Membrane Potentials
Acetylcholine
Ion Channels
Membranes

Keywords

  • Conductance
  • Gate
  • Ion selectivity
  • Reaction kinetics
  • Sulfhydryl

ASJC Scopus subject areas

  • Physiology

Cite this

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title = "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 α subunit",
abstract = "Ion channel function depends on the chemical and physical properties and spatial arrangement of the residues that line the channel lumen and on the electrostatic potential within the lumen. We have used small, sulfhydryl- specific thiosulfonate reagents, both positively charged and neutral, to probe the environment within the acetylcholine (ACh) receptor channel. Rate constants were determined for their reactions with cysteines substituted for nine exposed residues in the second membrane-spanning segment (M2) of the α subunit. The largest rate constants, both in the presence and absence of ACh, were for the reactions with the cysteine substituted for αThr244, near the intracellular end of the channel. In the open state of the channel, but not in the closed state, the rate constants for the reactions of the charged reagents with several substituted cysteines depended on the transmembrane electrostatic potential, and the electrical distance of these cysteines increased from the extracellular to the intracellular end of M2. Even at zero transmembrane potential, the ratios of the rate constants for the reactions of three positively charged reagents with αT244C, αL251C, and αL258C to the rate constant for the reaction of an uncharged reagent were much greater in the open than in the closed state. This dependence of the rate constants on reagent charge is consistent with an intrinsic electrostatic potential in the channel that is considerably more negative in the open state than in the closed state. The effects of ACh on the rate constants for the reactions of substituted Cys along the length of αM2, on the dependence of the rate constants on the transmembrane potential, and on the intrinsic potential support a location of a gate more intracellular than αThr244.",
keywords = "Conductance, Gate, Ion selectivity, Reaction kinetics, Sulfhydryl",
author = "Pascual, {Juan M.} and Arthur Karlin",
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T1 - State-dependent accessibility and electrostatic potential in the channel of the acetylcholine receptor

T2 - Inferences from rates of reaction of thiosulfonates with substituted cysteines in the M2 segment of the α subunit

AU - Pascual, Juan M.

AU - Karlin, Arthur

PY - 1998/6

Y1 - 1998/6

N2 - Ion channel function depends on the chemical and physical properties and spatial arrangement of the residues that line the channel lumen and on the electrostatic potential within the lumen. We have used small, sulfhydryl- specific thiosulfonate reagents, both positively charged and neutral, to probe the environment within the acetylcholine (ACh) receptor channel. Rate constants were determined for their reactions with cysteines substituted for nine exposed residues in the second membrane-spanning segment (M2) of the α subunit. The largest rate constants, both in the presence and absence of ACh, were for the reactions with the cysteine substituted for αThr244, near the intracellular end of the channel. In the open state of the channel, but not in the closed state, the rate constants for the reactions of the charged reagents with several substituted cysteines depended on the transmembrane electrostatic potential, and the electrical distance of these cysteines increased from the extracellular to the intracellular end of M2. Even at zero transmembrane potential, the ratios of the rate constants for the reactions of three positively charged reagents with αT244C, αL251C, and αL258C to the rate constant for the reaction of an uncharged reagent were much greater in the open than in the closed state. This dependence of the rate constants on reagent charge is consistent with an intrinsic electrostatic potential in the channel that is considerably more negative in the open state than in the closed state. The effects of ACh on the rate constants for the reactions of substituted Cys along the length of αM2, on the dependence of the rate constants on the transmembrane potential, and on the intrinsic potential support a location of a gate more intracellular than αThr244.

AB - Ion channel function depends on the chemical and physical properties and spatial arrangement of the residues that line the channel lumen and on the electrostatic potential within the lumen. We have used small, sulfhydryl- specific thiosulfonate reagents, both positively charged and neutral, to probe the environment within the acetylcholine (ACh) receptor channel. Rate constants were determined for their reactions with cysteines substituted for nine exposed residues in the second membrane-spanning segment (M2) of the α subunit. The largest rate constants, both in the presence and absence of ACh, were for the reactions with the cysteine substituted for αThr244, near the intracellular end of the channel. In the open state of the channel, but not in the closed state, the rate constants for the reactions of the charged reagents with several substituted cysteines depended on the transmembrane electrostatic potential, and the electrical distance of these cysteines increased from the extracellular to the intracellular end of M2. Even at zero transmembrane potential, the ratios of the rate constants for the reactions of three positively charged reagents with αT244C, αL251C, and αL258C to the rate constant for the reaction of an uncharged reagent were much greater in the open than in the closed state. This dependence of the rate constants on reagent charge is consistent with an intrinsic electrostatic potential in the channel that is considerably more negative in the open state than in the closed state. The effects of ACh on the rate constants for the reactions of substituted Cys along the length of αM2, on the dependence of the rate constants on the transmembrane potential, and on the intrinsic potential support a location of a gate more intracellular than αThr244.

KW - Conductance

KW - Gate

KW - Ion selectivity

KW - Reaction kinetics

KW - Sulfhydryl

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