TY - JOUR
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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U2 - 10.1085/jgp.111.6.717
DO - 10.1085/jgp.111.6.717
M3 - Article
C2 - 9607933
AN - SCOPUS:0031781630
SN - 0022-1295
VL - 111
SP - 717
EP - 739
JO - Journal of General Physiology
JF - Journal of General Physiology
IS - 6
ER -