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

Pascual , Juan M.; Karlin , Arthur

doi:10.1085/jgp.111.6.717

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Article| June 01 1998

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,

Juan M. Pascual

From the *Center for Molecular Recognition, and ‡Division of Pediatric Neurology, College of Physicians and Surgeons, Columbia University, New York 10032

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Arthur Karlin

From the *Center for Molecular Recognition, and ‡Division of Pediatric Neurology, College of Physicians and Surgeons, Columbia University, New York 10032

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Author and Article Information

Juan M. Pascual

From the *Center for Molecular Recognition, and ‡Division of Pediatric Neurology, College of Physicians and Surgeons, Columbia University, New York 10032

Arthur Karlin

From the *Center for Molecular Recognition, and ‡Division of Pediatric Neurology, College of Physicians and Surgeons, Columbia University, New York 10032

Address correspondence to Arthur Karlin, Center for Molecular Recognition, Columbia University, 630 West 168th Street, Box 7, New York, NY 10032. Fax: 212-305-5594.

2

We assume that AEAETS has a charge close to 2. If, for example, the pK_a of each amino group were the same as the minimum for MTSEA, 8.5, and they did not interact, then the average charge of AEAETS at pH 7.2 would be 1.9. If the ammonium groups did interact and, for example, if the first proton dissociated with a pK_a of 8 and the second with a pK_a of 9, then, at pH 7.2, the average charge would be 1.86.

3

The affinities of the thiosulfonates for the open channel of wild-type receptor are certainly lower than 10² M⁻¹, because millimolar concentrations resulted in no detectable reversible block of ACh-induced current. We do not know the affinity of any of the reagents for the “site” around a substituted Cys, but it is unlikely to be any greater than the affinity of the open wild-type channel.

4

The permeabilities of MTSEA and MTSET relative to K⁺ through the open ACh receptor were calculated from the shift in the reversal potential when the Na⁺ in oocyte bath solution was substituted by these reagents (Akabas et al., 1994a). The relative permeabilities were ∼0.6. Also, these reagents carried considerable inward current. By comparison, the permeabilities relative to Na⁺ of some organic cations of radii comparable to the radii of the cross-sections of MTSEA and MTSET are 0.36 for Tris, 0.87 for ethanolammonium, and 0.36 for N,N-dimethylethanolammonium (Cohen et al., 1992).

5

The reaction of the thiosulfonates with Cys in the channel, just as with thiols in solution, is with the negatively charged thiolate. This negative charge itself generates a negative electrostatic potential in the channel, which contributes to κ for the reaction of charged reagents. The ratio ρ, however, contains κ/k_ME for the charged reagent, in which the contribution of the Cys thiolate to ψ_S and κ should be approximately canceled by the contribution of the 2-mercaptoethanol thiolate to k_ME.

Received: December 31 1997

Accepted: March 12 1998

Online ISSN: 1540-7748

Print ISSN: 0022-1295

1998

J Gen Physiol (1998) 111 (6): 717–739.

https://doi.org/10.1085/jgp.111.6.717

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.

1998

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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

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